In Mexico and elsewhere in the Caribbean, the queen conch Strombus gigas is an endangered species. Understanding the genetic connectivity of their populations will support management strategies for long-term conservation of the species. Genetic diversity and population differentiation was assessed from samples collected at Banco Chinchorro and Isla Cozumel in the Mexican Caribbean and at Arrecife Alacranes in the Gulf of Mexico. Samples were obtained from the commercial capture at Banco Chinchorro (n = 50) and Isla Cozumel (n = 40) on March 2004. On November 2004, a non-invasive method for the Arrecife Alacranes sampling was applied, taking the hemolymph of live animals (n = 65) and releasing them to the wild. The mitochondrial DNA variation at two genes (COI and Cyt-b) was analyzed. Genetic diversity at the three locations ranged between 0.55-0.65 in COI and 0.87-0.94 in Cyt-b, showing no bottleneck evidences. A non-significant fixation index (F(ST) = 0.019, p = 0.161) and a Maximum Parsimony Network tree that did not show particular clades associated with any of the geographical locations, suggested a lack of statistically significant genetic differentiation among populations. Nevertheless, the cline patterns observed in both genetic diversity and haplotypic frequencies from Banco Chinchorro through Arrecife Alacranes, and the larger genetic distance between these locations from those between Isla Cozumel, Banco Chinchorro and Arrecife Alacranes, suggest the possibility of a pattern of isolation-by distance. The role of the main current systems over the potential genetic differences in S. gigas populations along the Mexican Caribbean, and the conservation management of S. gigas at these locations as discrete units is discussed.