Strawberry mottle virus (SMoV) is an important viral pathogen infecting strawberry (Fragaria spp.). The study was conducted to analyze the characterization of the molecular variation of SMoV and develop the methods for detection of SMoV by nested PCR and transcriptional enhancement techniques. The 3′ non-coding region (NCR) and large coat protein (LCP) gene of SMoV genome were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The specific segments were cloned and sequenced. The characterization of the molecular variation for some isolates of SMoV and phylogenetic analysis were studied. Based on the primers located in the conserved region of genome of SMoV, SMoV could be steadily detected using semi-nested PCR and transcriptional enhancement techniques. Both semi-nested PCR and transcriptional enhancement techniques were considerably more sensitive than the standard RT-PCR. The nucleotide sequences of NCR and partial LCP gene of Chinese isolates were obtained, and sequence analysis of the partial LCP gene of various SMoV isolates showed nucleotide identities ranging from 76.8 to 99.7%. There was a slight tendency for isolates to group according to their geographical origin. All 3 Polish isolates, 4 isolates of 7 Dutch isolates, and 3 isolates of 4 Chinese isolates formed a small separate branch, respectively. Two Germanic isolates had a far relationship with other isolates, and formed a separate clade. A high level of sequence variability was found among SMoV isolates, and the Germanic isolates were likely to a special strain group. The study was conducted to analyze the characterization of the molecular variation of SMoV and develop the methods for detection of SMoV by nested PCR and transcriptional enhancement techniques. The 3 ’non-coding region (NCR) and large coat protein (LCP) gene of SMoV genome were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The specific segments were cloned and sequenced. The characterization of the molecular variation for Some isolates of SMoV and phylogenetic analysis were studied. Based on the primers located in the conserved region of genome of SMoV, SMoV could be steadily detected using semi-nested PCR and transcriptional enhancement techniques. Both semi-nested PCR and transcriptional enhancement techniques were more sensitive than the standard RT-PCR. The nucleotide sequences of NCR and partial LCP gene of Chinese isolates were obtained, and sequence analysis of the partial LCP gene of various SMoV isolates showed nucleotide identities ranging from 76.8 to 99.7%. There was a slight tendency for isolates to group according to their geographical origin. All 3 Polish isolates, 4 isolates of 7 Dutch isolates, and 3 isolates of 4 Chinese isolates formed a small separate branch, respectively. Two Germanic isolates had a far relationship with other isolates, and formed a separate clade. A high level of sequence variability was found among SMoV isolates, and the Germanic isolates were likely to a special strain group.