Visual motility analysis is the basis for routine quality evaluation of stallion semen, although its prognostic value for fertilizing ability is considered low. The present study evaluated the ability of a novel computer-assisted motility analyzer (QualiSperm™) to determine the motility and velocity of ejaculated, extended stallion spermatozoa (collected from 10 stallions, 3 ejaculates/stallion) and following two different colloidal centrifugation methods (one- or twolayer), compared to visual evaluation by two independent operators. The Qualisperm™ instrument was able to retrieve and analyze ~10 times more spermatozoa per sample compared to routine visual estimation on the same time frame (~1,100 vs ~100 spermatozoa). The proportion of motile spermatozoa increased after the colloid-separation, compared to the extended ejaculates (P < 0.05) in some stallions. However, owing to the large variation seen among ejaculates and stallions, both for extended ejaculate (P < 0.05) as well as for the colloid centrifugations (P < 0.01), the differences were lost when the entire population was examined statistically. Interestingly, significant differences were seen for individual stallions between the measurements of Qualisperm™ and observers, as well as between observers (P < 0.05). Apart from the significantly higher number of spermatozoa analyzed at one time, the Qualisperm™ system provided a parameter that could simply not be estimated by visual assessment; mean sperm velocity (in µm/sec). Sperm velocity, upon which every computer assisted instrumentation base their evaluations, varied among stallions (and ejaculates within stallions, P < 0.05), with a tendency to increase after colloid-separation, thus suggesting the Qualisperm™ system might be able to differentiate sperm sub-populations. Due to its higher accuracy (in terms of sperm numbers examined) and speed, the Qualisperm™ system appears to be a suitable instrument for routine evaluation of equine semen