The distribution of the gopher tortoise tick (Amblyomma tuberculatum) has been considered intrinsically linked to the distribution of its primary host, gopher tortoises (Gopherus polyphemus). However, the presence of G. polyphemus does not always equate to the presence of A. tuberculatum. There is a paucity of data on the ecology, habitat preferences, and distribution of A. tuberculatum. The goals of this study were to assess the distribution of A. tuberculatum in southern Mississippi and to determine which, if any, habitat parameters explain the distribution pattern of A. tuberculatum. During 2006–2007, we examined 13 G. polyphemus populations in southern Mississippi for the presence of A. tuberculatum, and we measured a suite of habitat parameters at each site. Only 23% of the G. polyphemus populations supported A. tuberculatum, suggesting a more restricted distribution than its host. The results of our multivariate analyses identified several habitat variables, e.g., depth of sand and percentage of sand in the topsoil and burrow apron, as being important in discriminating between sites with, and without, A. tuberculatum. Amblyomma tuberculatum was only found at sites with a mean sand depth of >100 cm and a mean percentage of topsoil and burrow apron sand composition >94.0 and 92.4, respectively. Thus, environmental factors, and not just its host's range, seem to influence the distribution of A. tuberculatum.