There has been very limited use of computer assisted semen analysis (CASA) to evaluate reptile sperm. The aim of this study was to examine sperm kinematic variables in American crocodile (Crocodylus acutus) semen samples and to assess whether sperm subpopulations could be characterized. Eight ejaculates (two ejaculates/male) from four sexually mature captive crocodiles were obtained. An ISAS®v1 CASA-Mot system, with an image acquisition rate of 50 Hz, and ISAS®D4C20 counting chambers were used for sperm analyses. The percentages of motile and progressively motile spermatozoa did not differ among animals (P > 0.05) but there was a significant animal effect with regards to kinematic variables (P < 0.05). Principal component (PC) analysis revealed that kinematic variables grouped into three components: PC1, related to velocity; PC2 to progressiveness and PC3 to oscillation. Subpopulation structure analysis identified four groups (P < 0.05), which represented, on average, 9.8%, 32.1%, 26.8%, and 31.3% of the total sperm population. Males differed in the proportion of sperm in each of the kinematic subpopulations. This new approach for the analysis of reptile sperm kinematic subpopulations, reflecting quantifiable parameters generated by CASA system technology, opens up possibilities for future assessments of crocodile sperm and will be useful in the future development of assisted reproduction for these species.