To investigate the circulation of rabies virus in Ukraine, 78 rabies virus isolates were acquired from 14 states in 2002 and 2008-2010 for characterization. Partial sequences of nucleoprotein (359 nt) and glycoprotein (344 nt) genes were compared with those from neighbouring countries. The analysis identified 39 unique nucleoprotein genes and two geographically distinct RV variants belonging to the cosmopolitan lineage. The Ukrainian samples were similar to the North-East European lineage (NEE) (n = 19) and Russian group C (n = 20). The group C viruses were mainly isolated in Eastern Ukraine, from 9 regions, and from two other regions in Western Ukraine, suggesting the presence of group C throughout the country. These group C viruses are intermixed in bordering regions along the Dnieper River with viruses of group NEE, which were mainly isolated in six regions in Western Ukraine. Both nucleoprotein and glycoprotein gene analyses suggested evidence for cross-border movements of rabies virus.