Hygienic behavior is a social defense mechanism against parasites and pathogens in honeybees. We studied the genetic basis of hygienic behavior in African-derived Apis mellifera by performing RNA sequencing on brains of individual honeybee workers observed performing hygienic behavior, in order to identify expression changes linked with this behavior. We also used the transcriptome data to search for single nucleotide polymorphism (SNP) variation in genes previously associated with the trait in other A. mellifera populations. The analysis revealed 49 differentially expressed genes (DEG), most of them upregulated during hygienic acts. One DEG, Apidaecin, also showed SNP variation across samples, providing a promising candidate gene for both expression-based and heritable variation in hygienic behavior. We also identified 27 additional SNPs in the coding regions of five candidate genes previously linked with honeybee hygienic behavior. These results provide a useful starting place to analyze the genetic basis of hygienic behavior in African-derived honeybees.