A priority for sea turtle conservation is the identification of early biomarkers of environmental perturbation effects on the health of populations. The micronucleus (MN) assay is a noninvasive, sensitive, and economical in vivo assay for the determination of genotoxicity. It could help in ecotoxicological studies to determine if genotoxic chemicals are present in the environment and examine the chronic exposure of wildlife. The sample preparation protocol required to perform the MN assay in sea turtle peripheral blood is presented. The optimal times to fix (ethanol 80%) and stain (acridine orange) air-dried blood smears were evaluated. Acridine orange was used at a concentration of 0.01 g/100 ml in phosphate buffer (pH 7.4). MN and nuclear bud (NBUD) frequencies were determined in 1000 erythrocytes of free-ranging green sea turtles (Chelonia mydas) by using the species-specific developed protocol. The number of nuclear abnormalities in immature green turtles that inhabited 2 feeding grounds in the Mexican Caribbean was 0–17 NBUDs/1000 erythrocytes, and only 1 MN/1000 erythrocytes was detected in a single sea turtle specimen. Green turtles from the touristic and urbanized site presented a larger frequency of nuclear abnormalities (50%) than the value observed in the reference group. This protocol generates optimal results in a simple manner, and it is specific to validate and determine the frequency of spontaneous micronucleated erythrocytes in sea turtle peripheral blood. The preliminary results suggest the potential utility of the MN assay as biomarker of genotoxicity in sea turtles.