The two red algal species, Gracilaria birdiae Plastino and E.C. Oliveira and Gracilaria caudata J. Agardh, are the most important natural sources of agar in Brazil. Using the 454 sequencing system, we identified 464 and 487 perfect microsatellite loci in 6908 and 9602 sequences/contigs from G. birdiae and G. caudata, respectively. After a conservative removal of potentially problematic loci, 144 loci were tested (72 from each species). A total of 25 polymorphic microsatellite loci were defined (13 loci for G. birdiae and 17 loci for G. caudata, including 5 loci common to both species). The five microsatellite loci that cross-amplified in both species showed species-specific differences in allele size. Polymorphic microsatellite loci were used to assess the genetic diversity of both species in their main harvest and cultivation areas on the Brazilian coast. Gene diversity was similar in G. birdiae and G. caudata. However, significant heterozygote deficiency was observed in G. birdiae, whereas heterozygote excess occurred in G. caudata, suggesting that these two related species differ in their mating system. These results also raised new questions on their biology in the field and on their patterns of genetic structure across their geographical ranges. In addition, the 20 loci developed in this study proved successful in identifying each individual in the field as a unique multilocus genotype, and will be useful for studying lineage sorting, breeding programs, or conservation issues.