The nucleoprotein genes of Akabane virus S RNA segment from 21 Japanese and two Australian isolates were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) using a primer set containing the initiation and termination codon of the gene. The RT-PCR products were sufficiently produced from the purified virion RNAs of all the isolates, and then analyzed by enzymatic digestion with 11 restriction endonucleases. Digestion with the eight restriction enzymes revealed sequence variation of the isolates. Restriction fragment length polymorphism (RFLP) profiles obtained by digestion revealed the existence of four major groups (genogroups) among the isolates. The two Australian isolates had extremely different RFLP profiles than the Japanese isolates. The data demonstrate the usefulness of analyzing the RFLP patterns to understand the genetic variability of AKA virus isolated in Japan and Australia.