Abstract We have developed defined genetic lines of the hydroid Hydractinia symbiolongicarpus and confirmed earlier results showing that allorecognition is controlled by a single chromosomal region within these lines. In a large backcross population, we detected recombinants that display a fusibility phenotype distinct from typical fusion and rejection. We show that this transitory fusion phenotype segregates in a fashion expected of a single Mendelian trait, establishing that the chromosomal interval contains at least two genes that interact to determine fusibility. Using bulked segregant analysis, we have identified amplified fragment length polymorphisms (AFLP) cosegregating with fusibility, used these markers to independently confirm linkage of the two loci, and constructed a 3.4-cM map of an invertebrate histocompatibility complex.