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It is important to select the best honeybees that produce royal jelly to identify important molecular markers, such as major royal jelly proteins (MRJPs), and hence contribute to the development of new breeding strategies to improve the production of this substance. Therefore, this study focused on evaluating the genetic variability of mrjp3, mrjp5, and mrjp8 and associated allele maintenance during the process of selective reproduction in Africanized Apis mellifera individuals, which were chosen based on royal jelly production. The three loci analyzed were polymorphic, and produced a total of 16 alleles, with 4 new alleles, which were identified at mrjp5. The effective number of alleles at mrjp3 was 3.81. The observed average heterozygosity was 0.4905, indicating a high degree of genetic variability at these loci. The elevated FIS values for mrjp3, mrjp5, and mrjp8 (0.4188, 0.1077, and 0.2847, respectively) indicate an excess of homozygotes. The selection of Africanized A. mellifera queens for royal jelly production has maintained the mrjp3 C, D, and E alleles; although, the C allele occurred at a low frequency. The heterozygosity and FIS values show that the genetic variability of the queens is decreasing at the analyzed loci, generating an excess of homozygotes. However, the large numbers of drones that fertilize the queens make it difficult to develop homozygotes at mrjp3. Mating through instrumental insemination using the drones of known genotypes is required to increase the efficiency of Africanized A. mellifera-breeding programs, and to improve the quality and efficiency of commercial royal jelly apiaries.