The Mla locus in barley (Hordeum vulgare) conditions isolate-specific immunity to the powdery mildew fungus (Blumeria graminis f. sp. hordei) and encodes intracellular coiled-coil (CC) domain, nucleotide-binding (NB) site, and leucine-rich repeat (LRR)-containing receptor proteins. Over the last decades, genetic studies in breeding material have identified a large number of functional resistance genes at the Mla locus. To study the structural and functional diversity of this locus at the molecular level, we isolated 23 candidate MLA cDNAs from barley accessions that were previously shown by genetic studies to harbor different Mla resistance specificities. Resistance activity was detected for 13 candidate MLA cDNAs in a transient gene-expression assay. Sequence alignment of the deduced MLA proteins improved secondary structure predictions, revealing four additional, previously overlooked LRR. Analysis of nucleotide diversity of the candidate and validated MLA cDNAs revealed 34 sites of positive selection. Recombination or gene conversion events were frequent in the first half of the gene but positive selection was also found when this region was excluded. The positively selected sites are all, except two, located in the LRR domain and cluster in predicted solvent-exposed residues of the repeats 7 to 15 and adjacent turns on the concave side of the predicted solenoid protein structure. This domain-restricted pattern of positively selected sites, together with the length conservation of individual LRR, suggests direct binding of effectors to MLA receptors.