Sequence variation of the first internal transcribed spacer of ribosomal DNA (ITS-1) was examined and its application to the study of genetic variation was explored in four populations of farrer’s scallop Chlamys farreri. ITS-1 fragments,with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS-1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA (analysis of molecular variance) showed that the majority (96.26%) of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P< 0.05 (= 0.042), indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly. Sequence variation of the first internal transcribed spacer of ribosomal DNA (ITS-1) was examined and its application to the study of genetic variation was explored in four populations of farrer’s scallop Chlamys farreri. ITS-1 fragments, with a length of about 300 bp , of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS-1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA (analysis of molecular lar variance) showed that the majority (96.26%) of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P <0.05 (= 0.042), indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.