The Ryukyu five-lined skink (Plestiodon marginatus) is an island lizard that is even found in tiny islets with less than half a hectare of habitat area. We hypothesized that the island populations are maintained under frequent gene flow among the islands or independent of each other. To test our hypotheses, we investigated genetic structure of 21 populations from 11 land-bridge islands that were connected during the latest glacial age, and four isolated islands. Analyses using mitochondrial cytochrome b gene sequence (n = 67) and 10 microsatellite loci (n = 235) revealed moderate to high levels of genetic differentiation, existence of many private alleles/haplotypes in most islands, little contemporary migration, a positive correlation between genetic variability and island area, and a negative correlation between relatedness and island area. These evidences suggest a strong effect of independent genetic drift as opposed to gene flow, favoring the isolation hypothesis even in tiny islet populations. Isolation-by-distance effect was demonstrated and it became more prominent when the four isolated islands were excluded, suggesting that the pattern is a remnant of the land-bridge age. In a few island populations, however, the possibility of occasional overwater dispersals was partially supported and therefore could not be ruled out.