The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.