There are incomplete data about ant taxonomy and diversity in the Caribbean islands of Venezuela. In order to broaden myrmecological information in the Peri-Caribbean arid belt, ground-dwelling ant assemblage of the Macanao semi-arid peninsula (Margarita Island) was sampled with a Rapid Assessment Program (RAP). Two study sites on the northern slope of the peninsula were chosen, La Chica (LC) at 70 m of altitude and Cerro Macanao (CM) at 598 m of altitude. Sixty pitfall traps (30 per site) and 96 man-hours of manual collecting (48 man-hours per site) were employed. Overall, 40 species and 32 genera from eight subfamilies were recorded, twice the number of species previously reported for Margarita Island. Differences in ant species richness and structure were detected between both sites, with 31 species in CM and 25 species in LC. Such variations indicate that microclimatic conditions are more favorable in uplands than lowlands in Macanao. This situation may be attributed to the moisture gradient effect upon vegetation, and microclimatic conditions influenced by peninsular orography. Our findings corroborate that the pattern of higher diversity with higher altitude in arid zones also holds for the tropics, and suggest a continental origin for the Lesser Antilles ant fauna.