The black fly, Simulium ochraceum s.l., is believed to have reached San Cristobal Island, Galapagos prior to 1989, when reports of black fly biting began. The focus of this study is to establish the population structure of Simulium ochraceum s.l. on San Cristobal Island in order to determine if there has been a single or multiple introductions. We approached this problem by measuring the degree of heterozygosity and distribution in the collected samples. Enzymatic methods were used as a means of indirectly resolving genetic diversity among individuals and/or sites. The results denoted the possible presence of one or more polymorphisms in 60-70% of the individuals tested. As a more direct analysis, DNA from various individuals was sequenced and the results indicated polymorphisms in the majority of the individuals, many of which contained multiple polymorphic sites. Together, these results suggest either multiple introductions or a long-standing presence on the island.