This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials). PUBLICATION ABSTRACT