Lettuce (Lactuca sativa L.) belongs to the genus Lactuca L. and is an important vegetable worldwide. Over the past decades, there have been many controversies about the phylogeny of Lactuca species due to their complex and diverse morphological characters and insufficient molecular sampling. In this study we provide the most extensive molecular phylogenetic reconstruction of Lactuca, including African wild species, using two chloroplast genes (ndhF and trnL-F). The sampling covers nearly 40 % of the total endemic African Lactuca species and 34 % of the total Lactuca species. DNA sequences from all the subfamilies of Asteraceae in Genebank and those generated from Lactuca herbarium samples were used to establish the affiliation of Lactuca within Asteracaeae. Based on the subfamily tree, we selected 33 ndhF sequences from 30 species and 79 trnL-F sequences from 48 species to infer relationships within the genus Lactuca using randomized axelerated maximum likelihood and Bayesian inference analyses. Biogeographical, chromosomal and morphological character states were reconstructed over the Bayesian tree topology. We conclude that Lactuca contains two distinct phylogenetic clades—the crop clade and the Pterocypsela clade. Other North American, Asian and widespread species either form smaller clades or mix with the Melanoseris species. The newly sampled African endemic species probably should be treated as a new genus.