Comparison of GTG-banded cattle, sheep, goat, and fallow deer chromosomes showed conformity of G-banding patterns for chromosomes of fallow deer. In Bovidae species, autosomes were shown that were involved in centric fusion, giving a metacentric pair in fallow deer. Twenty-two microsatellite markers were amplified with the use of bovine specific primers in the investigated species. The results of cross-species amplification showed that microsatellites BM143, CSSM016 and TGLA53 seem to be conservative across all investigated ruminant species. Sequencing confirmed short tandem repeat motives of the TGLA53 marker among selected Bovidae and Cervidae species. About 60% of microsatellite loci were successfully amplified with the use of bovine primers in sheep and red deer species, 50% in goat, 55% in western roe deer, and 36% in fallow deer. This suggests that, on the DNA level, the genomes of red deer and sheep are more closely related to the bovine genome than genomes of the other studied ruminants.