Teeth are a classic model for studying vertebrate organogenesis and evolution. Despite the incredible phenotypic diversification of dentition in vertebrates, our understanding of the molecular and developmental basis behind this variation is limited. A derived benthic freshwater stickleback population has evolved a nearly two-fold increase in ventral pharyngeal tooth number compared to their ancestral marine counterparts. This evolved tooth gain provides an excellent system to study the developmental and molecular genetic basis of evolved dental variation. To ask when during development evolved tooth gain appears, we generated lab-reared developmental time courses of a low-toothed marine population and this high-toothed freshwater population. Early in development, no differences in dental patterning are observed. However, at late larval stages, an increase in tooth number, an increase in tooth plate area, and a decrease in tooth spacing arise. We identified genomic regions controlling these evolved patterning changes by mapping quantitative trait loci (QTL) controlling tooth number, area, and spacing in a marine by freshwater F2 cross. One large effect QTL controlling tooth number fine-maps to a genomic region containing an excellent candidate gene, Bone morphogenetic protein 6 (Bmp6). Stickleback Bmp6 is expressed in developing teeth, but no coding changes are found between the two populations. However, by quantitatively comparing allele specific expression of Bmp6, we find cis-regulatory changes have down-regulated the relative expression level of the freshwater Bmp6 allele at late, but not early, stages of development. To functionally test the role of Bmp6 in controlling tooth patterning, we generated predicted loss-of-function alleles of Bmp6 in freshwater sticklebacks. We found that Bmp6 is required for tooth formation and tooth plate area mirroring aspects of the evolved changes. Next, to discover enhancers that contain marine/freshwater sequence differences, we compared the chromosome 21 genomic sequences from fish with the tooth QTL to fish without the QTL. We identified a partially conserved region of the fourth intron of Bmp6 containing QTL-associated variants. This region is a tooth and fin enhancer that drives partially distinct expression patterns during tooth development compared to a 5’ Bmp6 tooth and fin enhancer we previously discovered that lacks consistent sequence differences associated with the tooth QTL. Future genetic and transgenic approaches will functionally test this intron 4 enhancer of Bmp6 as a candidate for underlying evolved tooth gain in sticklebacks.