Koala semen contains a heterogeneous mixture of sperm morphotypes, mainly attributable to extreme degree of shape variability displayed by the hooked sperm head. By analogy with other species, we anticipate that the morphotypes may exhibit correspondingly different sperm-motility behaviors, largely caused by the differences in hydrodynamic interactions with the suspending media. This trend has been shown in human spermatozoa where motility behavior was demonstrably correlated with the sperm head morphology (Overstreet et al. 1981). In this study, we have investigated the heterogeneity of koala sperm motility profiles in semen in an effort to determine whether distinct sperm subpopulations within ejaculates are recognizable by the use of computer-assisted sperm motility analysis. Ejaculates from 5 males were collected by electroejaculation, then diluted and transported in Tris-citrate-glucose (TCG) diluent. Spermatozoa were washed through a 35–60% Percoll gradient to separate seminal plasma and the majority of the prostatic bodies from spermatozoa. Spermatozoa from the washed pellet were then diluted in TCG at 35°C, incubated for 10 min, and video recorded using a negative phase ×10 objective. Sperm motion parameters were then analyzed using the Hobson sperm tracker (Hobson Vision Systems, UK: Holt et al. 1996 J. Androl. 17, 587–596). Multivariate pattern analysis (PATN; CSIRO Australia; Abaigar 1999 Biol. Reprod. 60, 32–41) was used to distinguish 3 sperm subgroups, consistently shown in each ejaculate, within the data (1936 tracks × 6 kinetic parameters; VCL, VAP, MAD, BCF, ALH, LIN). After group allocation by PATN, all parameters showed significant differences between each of the groups (P < 0.0001). Group 1, approximately 25% of the sperm tracks, showed profiles of spermatozoa with fast, non-linear motility (VCL 106.88 ± 28.15; BCF 3.23 ± 3.81; LIN 14.08 ± 10.20). Group 2, approximately 27% of sperm tracks, showed profiles of fast, linear motility (VCL 63.92 ± 13.50; BCF 7.90 ± 3.42; LIN 28.10 ± 12.15). Group 3, 48% of sperm tracks, showed profiles of slow, non-linear or circular patterns of motility (VCL 39.05 ± 11.92; BCF 0.02 ± 0.35; LIN 5.15 ± 4.88). The recognition of 3 clearly identifiable subgroups supports our hypothesis that heterogeneity of sperm motility patterns exists within koala ejaculates. These may be a reflection of the heterogeneity in sperm-head morphotypes in koala semen, but that remains to be investigated in more detail. The clear distinctions between these groups, and the observation that all 3 subpopulations exist in each of the ejaculates, also suggest that the spermatozoa exhibit functional differences, possibly related to biochemical or maturational status.