Overexploitation is the main anthropogenic threat to groupers (Epinephelidae) that aggregate to spawn. Fishing negatively affects their reproductive success and indirectly harms fishery economic yield. In the southern Gulf of Mexico, grouper catches, which include thirteen species, are in decline. A lack of biological information on each exploited species prevents optimising fishery management. Using histological examination of the gonads, the reproductive traits of red hind Epinephelus guttatus and yellowfin grouper Mycteroperca venenosa were studied from January 2008 to October 2009. Collections were made at two reef systems (Alacranes Reef and Bajos del Norte) on the continental shelf of the Yucatan Peninsula, Mexico, where these species form transient spawning aggregations. The results confirmed that previously identified spawning aggregation sites at both reefs constitute productive seasonal and perennial “hotspots” for both groupers; they spawn annually between January and April. Females of these protogynous hermaphroditic species exhibit a reproductive strategy characterised by asynchronous ovarian development organisation and ovulation. Sex ratios and maximum sizes at each reef suggest that populations of both groupers had a good conservation status as of the late 2000s. Both reefs are now marine protected areas, and a discussion is made of the consequent possible benefits to grouper population conservation and sustainability in the southern Gulf of Mexico.