MRI is commonly used to measure water’s diffusivity in opaque matter. In anisotropic tissues molecules tend to diffuse more freely along the principal axis of fibers than in the perpendicular planes [1]. To characterize such anisotropic molecular motions, diffusion tensor imaging (DTI) applies pulsed gradient echoes along multiple directions. Since DTI aims to describe the effects introduced by random molecular displacements spanning sub-mm ranges, ultrafast single-shot imaging techniques play key roles in this field. In most cases, and foremost in clinical applications, spin-echo echo planar imaging (SE EPI) is the method of choice for performing these rapid, diffusion-monitoring scans. However, SE-EPI can be challenged by eddy-current artifacts and by geometric distortions and ghosting effects related to field and chemical shift heterogeneities [2]. Recently, we have presented the diffusion-monitoring capabilities of an alternative single shot method, derived from spatiotemporal encoding (SPEN) principles [3]. Thanks to its reliance on a direct image acquisition that is not bound by k-space Nyquist sampling criteria, SPEN enables the use of stronger gradients along the low-bandwidth dimension, overcoming image distortions arising from B0-inhomogeneities and heterogeneous chemical environments [4-5]. The purpose of this study was to evaluate the usefulness of single-shot and interleaved SPEN methods to perform DTI under various pre-clinical and clinical settings. Additionally, the present study describes the formalism for analyzing SPEN DTI data, tailored to account for the spatially-dependent b-matrix weightings introduced by the sequence’s use of swept pulses acting in the presence of field gradients [7].To examine SPEN DTI’s performance, a number of systems were tested including ex vivo spinal cord and brain measurements, in vivo rodent brain mappings, and breast DTI scans of healthy volunteers. Phantom and rodents experiments were performed on an Agilent DD2® 7T/110mm pre-clinical horizontal magnet (Agilent Technologies, CA) using a quadrature-coil probe. Human breast DTI SPEN experiments were programmed and acquired at 3T on a Siemens TrioTIM® scanner (Erlangen, Germany), using a 4-channels bilateral breast receiver coil. This study compared SPEN and SE-EPI DTI results, from experiments including both single- and interleaved multi-shot acquisitions. Explicit calculations for the b-matrix elements were carried by programming scripts accounting for all gradient and pulse waveforms.To evaluate the usefulness of SPEN DTI analyses several SPEN strategies were adapted. The first (Figure 1A) incorporated an adiabatic 180˚ encoding pulse suitable for multi-slice imaging. A second sequence (Figure 1B) involved a 2D spatial/spatial pulse acting in unison with an encoding gradient Ge, progressively excites spins throughout the sample [6]. Figure 2 illustrates the eddy-currents effects for DTI pulse sequence using relatively short diffusion timings (δ = 3 ms, ∆ = 11 ms) yet relatively high bPGSE values (1200 s/mm²) reaching 43.2 G/cm. Ex-vivo coronal rat brain for SEMS (Fig. 2A) and SPEN based on a 180⁰ chirp encoding (Fig. 2B) showed similar anatomical information; for SE-EPI, by contrast, the fractional anisotropy (FA) maps were clearly distorted by the effects of eddy current interferences (Fig. 2C). These effects could not be avoided unless extending either δ or ∆, at the expense of longer echo times. Figure 3 presents diffusion properties measured for a human lactating breast via apparent diffusion coefficient (ADC) map (Fig. 3A) and FA map (Fig. 3B). A vector map representation of the ensuing principal diffusion eigenvectors (blue arrows in Fig. 3C) shows an alignment of the diffusivity along the anterior-posterior axis, reflecting the structure of milk ducts heading from the base of the breast toward the nipple. Thanks to SPEN’s robustness to field inhomogeneities, a closer look on this phenomenon is feasible towards the nipple of the breast–a region challenged by air/tissue interface problems.This work demonstrates the robustness of SPEN-based sequences enabling the implementation of DTI experiments with good sensitivity and resolution for brain scans and challenging environments like human breasts.Israel Science Foundation grant 795/13, the EU through ERC-2014-PoC grant # 633888, the Kimmel Institute for Magnetic Resonance and the generosity of the Perlman Family Foundation, are also acknowledged.