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American foulbrood (AFB) caused by Paenibacillus larvae is the most destructive honeybee bacterial disease and its dissemination via commercial bee pollen is an important mechanism for the spread of this bacterium. Because Mexico imports bee pollen from several countries, we developed a tRNA Cys -PCR strategy and complemented that strategy with MALDI-TOF MS and amplicon-16S rRNA gene analysis to evaluate the presence of P. larvae in pollen samples. P. larvae was not detected when the tRNA Cys -PCR approach was applied to spore-forming bacterial colonies obtained from three different locations and this result was validated by bacterial identification via MALDI-TOF MS. The genera identified in the latter analysis were Bacillus (fourteen species) and Paenibacillus (six) species. However, amplicon-16S rRNA gene analysis for taxonomic composition revealed a low presence of Paenibacillaceae with 0.3 to 16.2% of relative abundance in the commercial pollen samples analyzed. Within this family, P. larvae accounted for 0.01% of the bacterial species present in one sample. Our results indicate that the tRNA Cys -PCR, combined with other molecular tools, will be a useful approach for identifying P. larvae in pollen samples and will assist in controlling the spread of the pathogen.