Canine Parvovirus-2 (CPV-2) causes infectious viral disease characterized by severe diarrhea, vomiting, and high mortality, especially in young dogs. For routine laboratory diagnosis and molecular characterization of CPV-2, PCR analysis and phylogenetic analysis have been widely used methods in recent years. The aim of this study was to determine the molecular diagnosis and characterization of CPV-2 variants in puppies (0-6 months) with diarrhea symptoms and no vaccination against CPV-2 in the Central Anatolia region of Turkey. The phylogenetic analyses for VP2 gene were performed from the stools of the puppies (n = 50), which were found to be CPV-2 positive for the presence of nucleic acid by PCR, showing signs of gastroenteritis that were owned and cared for in the shelter environment. The results of the phylogenetic analysis, which included the study sequences of 4 each female and male puppies, indicated that both CPV-2a (4/8, 50%) and CPV-2b (4/8, 50%) variants were circulating in the study area. Molecular analyses also revealed that CPV-2 variants did not cause co-infection in these puppies. In conclusion, it can be stated that CPV-2a and CPV-2b variants continue to exist among the owned and stray dogs in the shelter in the Central Anatolia Region. Therefore, new vaccines should develop for successful fight against the infection and the vaccines should be prepared against CPV-2 virus antigenic types in different regions.