The feline panleukopenia virus (FPV) is a highly contagious virus that affects cats worldwide, characterized by leukopenia, high temperature and diarrhea. Recently, the continuous prevalence and variation of FPV have attracted widespread concern. The aim of this study was to investigate the isolation, genetic evolution, molecular characterization and epidemiological analysis of FPV strains among cats and dogs in China from 2019 to 2024. The 41 FPV strains, including 38 feline strains and 3 canine strains, were isolated from rectal swab samples by inoculating monolayer FK81 cells and performing a plaque purification assay. The viral and hemagglutination titers of these 41 FPV strains were 1010 TCID/0.1 mL and 7.0 log9.7 log, respectively. Based on the complete VP2 gene, the nucleotide homology of these FPV strains was 98.91100%, and the homology with 24 reference FPV strains from different countries and hosts was 98.85100%. The phylogenetic analysis revealed that 41 FPV strains were more closely related to the FPV strains of Asian origin (Asian FPV strain group) than those of European and American origin (European and American FPV strain group). Furthermore, 12 mutation sites of the VP2 protein were found in these FPV strains, of which 91 and 232 amino acid sites were previously reported. Moreover, the 91 amino acid site was found to be a positive selection site with the highest dN/dS value in the selection pressure analysis. Importantly, 35 FPV strains with 91S substitution in the VP2 protein (FPV-VP2-91S strains) had formed obvious evolutionary branches in the Asian FPV strain group. The analysis of all available VP2 protein sequences of Chinese FPV strains in the GenBank database showed that the occurrence rate of FPV-VP2-91S strains had been increasing from 15.63% to 100% during 2017~2024, indicating that the FPV-VP2-91S substitution in the VP2 protein was a noteworthy molecular characteristic of the dominant FPV strains in China. These results contribute to a better understanding of their genetic evolution and renew the knowledge of FPV molecular epidemiology.