AbstractABC transporters are the largest family of ATP-hydrolyzing transporters, with members in every sequenced genome, which transport substrates across membranes. Structural studies and biochemistry highlight the contrast between the global structural similarity of homologous transporters and the enormous diversity of their substrates. How do ABC transporters evolve to carry such diverse molecules and what variations in their amino acid sequence alter their substrate selectivity? We mutagenized the transmembrane domains of a conserved fungal ABC transporter that exports a mating pheromone and selected for mutants that export a non-cognate pheromone. Mutations that alter export selectivity cover a region that is larger than expected for a localized substrate-binding site. Individual selected clones have multiple mutations which have broadly additive contributions to specific transport activity. Our results suggest that multiple positions influence substrate selectivity, leading to alternative evolutionary paths towards selectivity for particular substrates, and explaining the number and diversity of ABC transporters.