A common evolutionary trend in highly selfing plants is the evolution of the "selfing syndrome", in which traits associated with pollinator attraction are lost or greatly reduced. Limited information is available on whether these trait reductions are favored by natural selection or result from reduced purifying selection coupled with genetic drift. This study attempted to distinguish between these two possibilities for the evolutionary loss of floral pigmentation in the highly selfing species Ipomoea lacunosa. This study also tested the hypothesis that loss of floral pigmentation is caused by downregulation or loss of function in a tissue-specific anthocyanin transcription factor, as has been found in other plants. F2 individuals of a cross between white and pigmented individuals revealed segregation at two epistatically acting loci: one affecting pigmentation in both corolla throat and limbs (Anl1) and one affecting limb pigmentation (Anl2). Individuals that are homozygous for the "white" allele at Anl1 have white throats and limbs regardless of genotype at Anl2. In individuals with pigmented throats, homozygosity of the "white" allele at Anl2 produces white limbs. Flower color variation at Anl1 cosegregates with an R2R3-Myb anthocyanin transcription factor, which is down-regulated in white-flowers but not in pigmented flowers. Differential expression of the two alleles of this gene indicates that down regulation is caused by a cis-regulatory change. Finally, allele-frequency differences at Anl1 were substantially and significantly greater than differences in allele frequencies at four microsatellite loci. These results are consistent with the hypotheses that the identified R2R3-Myb gene corresponds to Anl1 and that evolutionary loss of pigmentation in I. lacunosa was caused by selection. They are also consistent with previous studies demonstrating that loss of floral pigmentation is usually caused by down-regulation or functional inactivation of an R2R3-Myb gene.