A study was carried out to assess the quality of frozen-thawed semen using the sex separation method and the traditional method in a comparative aspect, and the authors believe that the work will be of some interest to specialists in zootechnical and biological fields, as well as to veterinary specialists. The work was carried out at the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst and on the basis of LLC “Agrofirma Zarya”, Bogorodsky district, Nizhny Novgorod region in the period from 2017 to 2022. usng the semen of eight sires. To assess the quality of the semen, the index of nuclear DNA fragmentation (nDNA) in chromatin was determined, and the activity of spermatozoa was assessed visually and using the Biola AFS-500 sperm analyser (ZAO Biola, Moscow). On the basis of the studies conducted, it was found that the morphology of the spermatozoa depends on the individual characteristics of the sires; the content of abnormal spermatozoa varies from 2 to 8% when collected by the traditional method and from 4.10 to 15% when collected by the sexing method. A significant difference between the sires is observed in the iDNA fragmentation index – this indicator varies from 2.66 to 8.62% and from 8.50 to 28.57%, respectively. The division of spermatozoa by sex has a direct effect on the quality indicators, leading to a decrease in their activity. This indicator is 11.2% higher in semen obtained by the traditional method than by the alternative technique. The number of abnormal cells in the semen divided by sex is 2.2% higher than in the semen collected by the traditional method, and the proportion of spermatozoa with fragmented iDNA is 7% higher. The velocity of spermatozoa in the studied doses of semen collected using the technique of sex separation was 96–113 µm/s immediately after thawing, and five hours after incubation at +38°C in a thermostat this indicator decreased to the level of 20–34 µm/s. The age of puberty of their calves obtained from the sexed semen from birth to fruitful insemination is 490 days, which is 16 days more than in group I, where heifers were obtained from the semen frozen in the traditional way (TS). The term of fruiting in heifers born from the use of the sexed semen (SS) was 273 days, while that of heifers born from the use of traditionally frozen semen (TS) was 275 days (2 days more). The age of heifers from birth to first calving in the SS group was 762 days and was 14.4 days more than in the TS group, which was 748 days.