[Objetive] The objective of the present study was to characterize different sperm subpopulations based on morphometric parameters of frozen-thawed semen in two bovine subspecies using a CASA system. [Methodology] The experiment was carried out at the Costa Rica Institute of Technology from May to December 2023. Spermatozoa from 10 bulls (five animals of each species Bos taurus and Bos indicus) were evaluated after thawing of the semen doses by an ISAS^®v1, computer-assisted sperm analysis (CASA)-Morph system. Sub-populations of head morphometric spermatozoa were characterized using multivariate procedures such as principal components (PCs) analysis and clustering methods (k-means model) [Results] The ejaculate of male exhibits significant heterogeneity and comprises diverse sperm subpopulations with differing morphometric patterns. Three different sperm subpopulations were identified from three PCs: t head size, head shape, and symmetry of the sperm head and the degree to which it was pyriform. The proportions of the different sperm subpopulations varied in the two-bovine subspecies; Bos taurus and Bos indicus. Results indicated that subpopulations SP1, SP2, and SP3 were different for PC criteria and these differences were relevant. The variability in sperm morphometry assessments underscores the need to standardize semen evaluation protocols [Conclusions] These findings highlight the importance of knowing the diversity in sperm morphometry between subspecies to both improve in vitro evaluation tests and to distinguish subspecies.