The identification of genomic regions with sufficient variation to elucidate fine-scale relationships among closely related species is a major goal of phylogenetic systematics. However, the accumulation of such multi-locus data sets brings its own challenges, given that gene trees do not necessarily represent the true species tree. Using genomic tools developed for Solanum (Solanaceae), we have evaluated the utility of nuclear conserved ortholog set II (COSII) regions for phylogenetic inference in tribe Lycieae (Solanaceae). Five COSII regions, with intronic contents ranging from 68% to 91%, were sequenced in 10 species. Their phylogenetic utility was assessed and compared with data from more commonly used nuclear (GBSSI, nrITS) and cpDNA spacer data. We compared the effectiveness of a traditional total evidence concatenation approach versus the recently developed Bayesian estimation of species trees (BEST) method to infer species trees given multiple independent gene trees. All of the sampled COSII regions had high numbers of parsimony-informative (PI) characters, and two of the COSII regions had more PI characters than the GBSSI, ITS, and cpDNA spacer data sets combined. COSII markers are a promising new tool for phylogenetic inference in Solanaceae, and should be explored in related groups. Both the concatenation and BEST approaches yielded similar topologies; however, when multiple individuals with polyphyletic alleles were included, BEST was clearly the more robust approach for inferring species trees in the presence of gene tree incongruence.