Abstract Sperm cryopreservation is one of the most important elements for the creation of genetic material cryobanks in order to preserve the gene pool of poultry. Cryopreservation methods and parameters directly affect the viability of germ cells after thawing. The effect of freeze-thaw cycles on biological usefulness of bird sperm was studied. Semen was frozen in paillettes. Thawing sperm was carried out at a temperature of 38 °С. Sperm activity was assessed using CASA technology “ARGUSSOFT”. Sperm motility after cryopreservation decreased in roosters, quails and guinea fowls by 62 ± 3 %, 66 ± 1 % and 60 ± 1 %, respectively. The proportion of live sperm also decreased: in roosters - from 89 ± 4 % to 48 ± 2 %, in quails - from 93 ± 3 % to 49 ± 3 %, in the guinea fowls - from 92 ± 2 % to 45 ± 4 %. As a result of freezing and thawing, the proportion of spermatozoa with abnormal morphology increased. A change in the frequency of anomalies occurrence in individual segments was observed. The number of spermatozoa with flagella pathology was increased. The proportion of sperm with pathology of the head, middle section and flagellum increased by 0.4 %, 0.4 % and 1.3 % (P ≤ 0.001) respectively, in the frozen-thawed samples of roosters, compared with the indicators established for a freshly obtained ejaculate. A similar trend was observed in other poultry types. Thus, the freeze-thaw cycle had a negative effect on the activity and viability of poultry spermatozoa. Supported by RSF No 16-16-04104.