ABSTRACT The genetic basis of phenotypic differences between species is a longstanding question in evolutionary biology. How new genes form and selection acts to produce differences across species are fundamental to understanding how species evolve. Adaptation and genetic innovation arise in the genome from a variety of sources. Functional genomics requires both genetic discoveries and empirical testing to observe adaptation between lineages. We explore two species of Drosophila from the island of São Tomé and mainland Africa, D. santomea and D. yakuba . These two species have varying distributions based on elevation on São Tomé, with populations of D. yakuba also inhabiting mainland Africa. Genomic/genetic evidence shows genes between species may have a role in adaptation to higher UV tolerance. We conducted empirical UV assays between D. santomea and both D. yakuba populations. Flies were shocked by UVB radiation for 30 min on a transilluminator apparatus. Custom 5‐wall acrylic enclosures were constructed for viewing and containment of flies. Island groups show significant differences between fall‐time under UV stress and recovery time post‐UV stress test between populations and by sex. This study shows evidence that mainland flies are less resistant to UV radiation than their island counterparts. Differential expression analysis also shows potential for new mutations and local adaptation for DNA repair of D. santomea . Understanding the mechanisms and processes that promote adaptation and testing traits within the context of the genome is crucially important to understand evolutionary machinery.