Learning of most motor skills is constrained in a species-specific manner. However, the proximate mechanisms underlying species-specific learned behaviors remain poorly understood. Songbirds acquire species-specific songs through learning, which is hypothesized to depend on species-specific patterns of gene expression in functionally specialized brain regions for vocal learning and production, called song nuclei. Here, we leveraged two closely related songbird species, zebra finch, owl finch, and their interspecific first-generation (F1) hybrids, to relate transcriptional regulatory divergence between species with the production of species-specific songs. We quantified genome-wide gene expression in both species and compared this with allele-specific expression in F1 hybrids to identify genes whose expression in song nuclei is regulated by species divergence in either cis- or trans-regulation. We found that divergence in transcriptional regulation altered the expression of approximately 10% of total transcribed genes and was linked to differential gene expression between the two species. Furthermore, trans-regulatory changes were more prevalent than cis-regulatory and were associated with synaptic formation and transmission in song nucleus RA, the avian analog of the mammalian laryngeal motor cortex. We identified brain-derived neurotrophic factor (BDNF) as an upstream mediator of trans-regulated genes in RA, with a significant correlation between individual variation in BDNF expression level and species-specific song phenotypes in F1 hybrids. This was supported by the fact that the pharmacological overactivation of BDNF receptors altered the expression of its trans-regulated genes in the RA, thus disrupting the learned song structures of adult zebra finch songs at the acoustic and sequence levels. These results demonstrate functional neurogenetic associations between divergence in region-specific transcriptional regulation and species-specific learned behaviors.