The sequences of internal transcribed spacers of ribosomal DNA (ITS rDNA) are widely used to study the species relationships in different groups of organisms, yet they have not been applied to the study of benthic foraminifera until now. Here, we use the ITS rDNA sequences to examine the phylogenetic relationships between two morphospecies, Planoglabratella opercularis and Planoglabratella nakamurai, collected from around the Japanese Islands. Analysis of 27 ITS rDNA sequences shows that P. opercularis is composed of two genetically distinct species, A and B, the latter one being more closely related to P. nakamurai than to P. opercularis type A. Detailed morphological study confirms our molecular data, which reveals some typical P. nakamurai features in P. opercularis type B. Geographic distribution of both genotypes was examined by using PCR-based RFLP method. Analysis of 58 specimens and sequenced data collected in 10 locations shows that P. opercularis type A is widely distributed throughout the Japanese Islands, while P. opercularis type B is present only in a limited area in the central part of Honshu Island, where it coexists with P. nakamurai and P. opercularis type A. This genetic, morphologic, and geographic evidence suggests that P. opercularis type B is a separate species. Planoglabratella opercularis type B and P. nakamurai are the sister groups of P. opercularis type A. Our study shows that ITS rDNA can be a useful tool for genetic distinction of cryptic species in benthic foraminifera and for studying their intraspecific variation.