We determined the groESL sequences of three species of nutritionally variant streptococci (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and three Gemella species (Gemella morbillorum, Gemella haemolysans, and Gemella sanguinis). The nucleotide sequence similarities between the groES and groEL genes of the above genera were 41.7 to 85.9% and 63.7 to 84.3%, respectively. The intraspecies similarities of groESL sequences for the isolates of Abiotrophia and Granulicatella species were 94.4 to 97.8% for groES and 94.0 to 98.2% for groEL. For Ge. morbillorum and Ge. sanguinis, all strains showed the same groESL spacer length (8 bp), and sequence identities within species were >97.8% for groES and >96.1% for groEL. However, higher intraspecies heterogeneity was observed in Ge. haemolysans. Phylogenetic analysis of groEL sequences separated the 6 isolates of Ge. haemolysans into two subgroups. Among these isolates, three isolates with the same groESL spacer region length (45 bp) clustered together but were distant from the ATCC reference strain (with a spacer length of 8 bp). The remaining three isolates, with a spacer length of 50 or 8 bp, clustered together. Although 16S rRNA gene sequence analysis did not provide enough discrimination for the 6 Ge. haemolysans isolates, rpoB gene sequence analysis supported the subgrouping. Based on the obtained groESL sequences, we developed a multiplex PCR that enables simple, rapid, and accurate identification of Abiotrophia, Granulicatella, and Gemella at the genus level. This assay would be helpful for identifying these fastidious and slow-growing organisms in clinical laboratories.