It has been argued that the common ancestor of about 75% of all dicots possessed an S-RNase-based gametophytic self-incompatibility (GSI) system. S-RNase genes should thus be found in most plant families showing GSI. The S-RNase gene (or a duplicate) may also acquire a new function and thus genes belonging to the S-RNase lineage may also persist in plant families without GSI. Nevertheless, sequences that belong to the S-RNase lineage have been found in the Solanaceae, Scrophulariaceae, Rosaceae, Cucurbitaceae, and Fabaceae plant families only. Here we search for new sequences that may belong to the S-RNase lineage, using both a phylogenetic and a much faster and simpler amino acid pattern-based approach. We show that the two methods have an apparently similar false-negative rate of discovery (approximately 10%). The amino acid pattern-based approach produces about 15% false positives. Genes belonging to the S-RNase lineage are found in three new plant families, namely, the Rubiaceae, Euphorbiaceae, and Malvaceae. Acquisition of a new function by genes belonging to the S-RNase lineage is shown to be a frequent event. A putative S-RNase sequence is identified in Lotus, a plant genus for which molecular studies on GSI are lacking. The hypothesis of a single origin for S-RNase-based GSI (before the split of the Asteridae and Rosidae) is further supported by the finding of genes belonging to the S-RNase lineage in some of the oldest lineages of the Asteridae and Rosidae, and by Baysean constrained tree analyses.