Electrophoretic analyses of 14 kinds of enzymes and two kinds of blood proteins were made in 525 toads of 40 populations of Bufo japonicus distributed widely in Japan. These populations belong to five subspecies, B. j. montanus, B. j. japonicus (including B. j. formosus and B. j. hokkaidoensis), B. j. torrenticola, B. j. yakushimensis and B. j. miyakonis. The enzymes and blood proteins are controlled by genes at 22 loci. The fixation indexes calculated according to WRIGHT (1978) showed that the Miyako population of B. j. miyakonis is remarkably differentiated from the other 39 populations belonging to four subspecies. The rates of average heterozygosity were 1.0∿21.7%, 10.70n the average. The proportions of polymorphic loci in the 40 populations were 5∿65%, 36.00n the average. The mean numbers of alleles per locus were 1.05∿1.95,1.46 on the average. Examination of the geographic distribution of alleles at the 22 loci in the 40 populations of B. japonicus showed that there are distinct gradients from east to west in the alleles at the AAT-A, IDH-B, LDH-B, ME-B and MPI loci. Some of the alleles at the Ab and Hb loci are abundantly gathered in the populations distributed in some districts. At each of the 15 loci other than the foregoing seven, one of the alleles is overwhelmingly abundant in all the 40 populations or the 39 populations excluding the Miyako population which is exclusively or partly occupied by another allele. The genetic relationships among the 40 populations of Bufo japonicus distributed in Japan were conjectured by estimating the genetic distances by the method of NEI (1975) and a dendrogram was drawn using the UPGMA clustering method. It was found that B. japonicus has evolved into the eastern and western groups, except that B. j. miyakonis seems to have come from the mainland of eastern Asia.