Lateral genetic transfer (LGT) is an important adaptive force in evolution, contributing to metabolic, physiological and ecological innovation in most prokaryotes and some eukaryotes. Genomic sequences and other data have begun to illuminate the processes, mechanisms, quantitative extent and impact of LGT in diverse organisms, populations, taxa and environments; deep questions are being posed, and the provisional answers sometimes challenge existing paradigms. At the same time, there is an enhanced appreciation of the imperfections, biases and blind spots in the data and in analytical approaches. Here we identify and consider significant open questions concerning the role of LGT in genome evolution.