Allorecognition is the capability of an organism to recognize its own or related tissues. The colonial ascidian Botryllus schlosseri, which comprises five genetically distinct and divergent species (Clades A-E), contains two adjacent genes that control allorecognition: fuhc(sec) and fuhc(tm). These genes have been characterized extensively in Clade A and are highly polymorphic. Using alleles from 10 populations across the range of Clade A, we investigated the type and strength of selection maintaining this variation. Both fuhc genes exhibit higher within-population variation and lower population differentiation measures (F-sT) than neutral loci. The fuhc genes contain a substantial number of codons with >95% posterior probability of d(N)/d(s) >1. fuhc(sec) and fuhc(tm) also have polymorphisms shared between Clade A and Clade E that were present prior to speciation (trans-species polymorphisms). These results provide robust evidence that the fuhc genes are evolving under balancing selection. (C) 2016 Elsevier Ltd. All rights reserved.