Native American mitochondrial DNA belongs to one of ve haplogroups de ned by lineage-speci c markers. Haplogroup frequency distribution is non-random among cultures. At historical contact, the Gabrielino occupied the southernmost California Channel Islands and the adjacent Los Angeles Basin. The Chumash thrived on the northern Channel Islands and Santa Barbara mainland. The Gabrielino were linguistically, culturally, and possibly genetically distinct from the Chumash. Haplogroup frequencies were determined for two prehistoric populations from San Clemente Island (Eel Point and the Nursery Site) to investigate Uto-Aztecan migration onto the southern Channel Islands. Analysis included three measures of genetic distance and phylogenetic analysis, as well as Fisher’s exact tests. The Eel Point and Nursery Site frequency distributions were compared to one another, and to extant Uto-Aztecan and Great Basin/California populations. Results suggest that the prehistoric occupants of Eel Point and the Nursery Site were not closely related to one another or to the Chumash.