Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte quality and competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is essential to employ superior methods of selecting high quality sperm to maximize IVP outcomes. Clinical sperm selection techniques for stallions are generally limited to swim-up (SU), density gradient centrifugation (DGC), density gradient-swim up combination (DG-SU), and variations of these methods, despite the rapidly expanding use of novel techniques in humans and other species. One novel method of sperm selection is a microfluidic (MF) chip, which utilizes natural rheotactic behaviors of sperm to select for a highly motile and viable population of cells that would be optimal for IVP. However, no comprehensive comparison between MF and the conventional methods of DGC and DG-SU has been performed for stallions. Thus, we aimed to compare conventional equine sperm selection techniques to the novel method of MF sorting. Ejaculates (n = 22) were partitioned into fresh (∼23°C, 0 h; n = 12) and cooled (∼4°C, 24 h; n = 10) groups, and processed by wash-only, SU, DGC, DG-SU, and MF (ZyMōt™ Multi, 850µL, Sperm Separation Device™, DxNow, Gaithersburg, MD). Sperm motility, progressive motility, viability, and normal morphology,parameters used as basic estimates of sperm quality, were evaluated for both the unprocessed fraction and post-selected fractions. Among the compared methods of sperm selection, MF was user-friendly and effective in selecting high quality sperm as determined by individual parameters and by a summative sperm quality index (SQI) that combined individual factors. MF selected sperm with greater percentages of motility, progressive motility, viability, and normal morphology than unprocessed, wash-only, and SU-selected groups (P < 0.0001). Additionally, sperm selected by MF had numerically greater motility, progressive motility, and SQI, in comparison to DGC and DG-SU groups (P > 0.05). Cell viability and morphology parameters were comparable among MF, DGC, and DG-SU. Thus, MF-selected sperm not only exhibited numerically higher quality than DG-SU and DGC-selected sperm, but also the method did not use potentially harmful centrifugation steps. Therefore, MF may pose additional advantages over conventional methods. In conclusion, to select for superior sperm within a total ejaculate, implementation of MF technologies could be of interest to equine ICSI laboratories. This study was funded by the Theriogenology Foundation and the Center for Equine Health at UC Davis.