Our newly designed primers were evaluated for the molecular analysis of specific groups of the sqr gene encoding sulfide : quinone reductase (SQR) in sediment environments. Based on the phylogenetic analysis, we classified the sqr sequences into six groups. PCR primers specific for each group were developed. We successfully amplified sqr-like gene sequences related to groups 1, 2 and 4 from diverse sediments including a marine sediment (SW), a tidal flat (TS), a river sediment (RS) and a lake sediment (FW). We recovered a total of 82 unique phylotypes (based on a 95 % amino acid sequence similarity cutoff) from 243 individual sqr-like gene sequences. Phylotype richness varied widely among the groups of sqr-like gene sequences (group 1>group 2>group 4) and sediments (SW>TS>RS>FW). Most of the sqr-like gene sequences were affiliated with the Proteobacteria clade and were distantly related to the reference sqr gene sequences from cultivated strains (less than approximately 80 % amino acid sequence similarity). Unique sqr-like gene sequences were associated with individual sediment samples in groups 1 and 2. This molecular tool has also enabled us to detect sqr-like genes in a sulfur-oxidizing enrichment from marine sediments. Collectively, our results support the presence of previously unrecognized sqr gene-containing micro-organisms that play important roles in the global biogeochemical cycle of sulfur.