The use of multiplex PCR assays to screen microsatellite variation is a rapid, economically efficient and robust approach to acquire population genetic data. To date, however, few such multiplex assays have been reported in large-scale studies of a fishery. We developed a two reaction, 12 locus microsatellite assay for brown trout, one of the most important aquaculture species in Europe. We describe the steps taken to choose loci that provide unambiguous data in an applied fisheries context. Several parameters were tested for assay optimisation, among which the relative concentration of each primer pair and the PCR elongation time and temperature were the most important. The assay was optimised for the two lineages of brown trout (Atlantic and Danubian) found in Austria. Evaluation on additional lineages of Salmo trutta as well as other Salmo species showed that with few adjustments the assay could be reliably used in all lineages of brown trout as well as S. marmoratus, S. ohridanus, and S. obtusirostris, but probably not for S. salar without major change. (c) 2006 Elsevier B.V. All rights reserved.