Flavonoids are important secondary metabolites that play an integral role in protecting plants against UV radiation and other forms of environmental stress. Given widespread impacts of environmental effects associated with latitude on a multitude of biological systems and a well-documented increase in solar radiation towards the equator, plant flavonoid production is expected to increase as a response to factors associated with decreasing latitude. Using data from a Neotropical genus (Ruellia) that spans an exceptionally broad latitudinal gradient, we tested a hypothesis of a positive latitudinal gradient in flavonoid concentration and assessed other factors that influence flavonoid production including habitat type (xeric vs. wet), altitude, phylogenetic relatedness, and pleiotropic effects. Two flavones with peak absorbance in ultraviolet wavelengths, apigenin and luteolin, were detected across all species. Transcriptome data confirm high expression of the gene required for flavone biosynthesis, flavone synthase (FNS). Contrary to our prediction, data revealed a positive correlation between flavone concentration and higher latitudes. Further, we recovered strong impacts of xeric habitat, pleiotropy, and phylogenetic relatedness on flavone concentrations. This study documents a complex interplay of ecological, historical, phylogenetic relatedness, and pleiotropic factors driving plant flavonoid production.