An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003-2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.