1. The watercourses of Austria are mainly part of the Danube drainage system. Nonetheless, only few brown trout (Salmo trutta) populations of the Danubian phylogenetic lineage have been found in this region, not being introgressed by hatchery-reared fish of the allochthonous Atlantic lineage. The present study was aimed at identifying further waterbodies that might harbour the non-admixed gene pool of the Danubian clade trout.
2. In a first step, the complete mitochondrial DNA control region of 447 specimens from 20 sampling sites was sequenced to screen for the presence of specific haplotypes. In a second step, 332 individuals from 15 populations were analysed at 10 microsatellite DNA loci.
3. Although sampling concentrated on remote Alpine headwater streams, introgression of hatchery strains was more common than expected from existing stocking records. For seven populations, however, no evidence of recent hatchery trout introgression was found and it is suggested that they might represent the indigenous gene pool.
4. Populations showing no evidence of hatchery introgression displayed lower levels of intrapopulation variability and higher degrees of differentiation compared to purported admixed populations.
5. Our study demonstrates the necessity of combining different molecular markers to reveal the impact of introgression into wild populations, since at some sampling sites admixture would have remained undetected if only a single molecular technique was applied.
6. The identification and characterisation of non-introgressed populations provides important information for setting conservation priorities and preventing further loss of genetic integrity of a unique element of the native fish fauna.