Analyses of 11 isoenzyme loci of Ochlerotatus eatoni (Edwards, 1916), endemic to two Macaronesian Islands (Madeira and Tenerife, Canary Islands), revealed substantial genetic structure in the study populations. Samples from sites on the south and north of Madeira displayed a significant reduction of variability compared with those from central Madeira and Tenerife. The Tenerife population exhibited a severe deficit of heterozygosity with similar magnitude across all the loci examined. The complex pattern of variation in Oc. eatoni is because of interplay of breeding structure, genetic drift, and geographical and historical factors. From these findings, we concluded that island colonization by Oc. eatoni was not marked by founder effect.