The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual thresholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32-113, 61-105, 48-104 and 29-91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56-94.59%, 93.92-97.02%, 76.14-97.76% and 79.45-83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.