The evolution of flower color in the genus Nicotiana has been dependent on the presence of various pollinators. Different colored flowers are created by pigments of different hues, which are produced by the branched pattern of the flavonoid biosynthetic pathway. The objective of this study is to identify the genetic basis of flower color shifts in Nicotiana by determining which evolutionary pressures are present on flavonoid biosynthetic pathway genes. We obtained available transcriptome data and extracted the sequences of the flavonoid biosynthetic pathway genes. Sequences of each gene were then used to infer phylogeny and analyses were run to determine the ratio of the rate of nonsynonymous mutations to the rate of synonymous mutations (dN/dS). This ratio will indicate which evolutionary pressure is acting on the flavonoid biosynthetic pathway genes including: purifying selection (dN/dS 1). Overall, we observed that the flavonoid biosynthetic pathway genes tended to be under purifying selection. We hypothesized that species that do not produce anthocyanins will be under more relaxed purifying selection in comparison to those that do, suggesting that there is less pressure to maintain the original sequence as the genes encoding for anthocyanins are not being used by these species. In flower color comparisons between species without anthocyanins versus those with anthocyanins, we saw significantly different dN/dS ratios for the genes DIHYDROFLAVONOL 4-REDUCTASE (DFR), FLAVONOID 3’-HYDROXYLASE (F3’H), and FLAVONOID 3’,5’-HYDROXYLASE (F3’5’H). For the genes F3’H and F3’5’H, we observed relaxed purifying selection acting on the species without anthocyanins, supporting our original hypothesis. However, for the gene DFR, we observed relaxed purifying selection acting on the species with anthocyanins, contradictory to our original hypothesis. We found a frameshift mutation resulting from a one base pair deletion in the N. sylvestris homeolog of DFR in some N. tabacum accessions, which likely contributes to the relaxed purifying selection seen in species producing anthocyanins. Based on their placement at branching points in the pathway, it is unsurprising that DFR, F3’H, and F3’5’H have significantly different dN/dS ratios in species with and without anthocyanins since mutations in these genes may alter the flux of the pathway to produce different pigments. When simulating the evolution of the pathway using computational models, the mutations that accumulated to produce flower color shifts were concentrated in the genes at the branches of the pathway, suggesting the corroboration of simulated and empirical data.