We applied the theory of random genetic drift to determine the divergence history of a closed cattle population over the relatively short timescale of several hundred years. The divergence history of the closed population of Mishima Island cattle, a national natural treasure of Japan, was examined, and the results were compared with historical documents. Inbreeding depression in the isolated population was investigated for body size and fertility. Twenty-one DNA microsatellite markers in Mishima Island cattle and 3 major breeds from the mainland were genotyped. For the mainland breeds, all 21 or 20 markers were segregating. However, nearly half the number of loci (9 of 21) was fixed in the Island cattle. The average number of alleles per locus of Island cattle was markedly lower than that in the mainland breeds. These results support the theory that Island cattle have been isolated for a considerable period of time. The number of generations of isolation was estimated as 14.1-22.6, and the year of divergence was calculated as 1778-1846. In view of these findings, we propose that Island cattle diverged from the mainland population at around 1800 and were isolated for about 200 years. These estimates are in agreement with historical documents showing that divergence occurred between 1672 and 1880. The total inbreeding coefficient of the present population was predicted to be in the range of 0.51-0.60. However, historical reports dated over 100 years do not support changes in fertility, so that there is no evidence for inbreeding depression.