Rapid and accurate sex-identification of salmonids has remained an important challenge for aquaculture and wild population study and management. A molecular method looking for the presence of the male-specific sdY gene has recently been proposed. However, the size of the amplified fragment (similar to 500pb) and the DNA revealing technique (agarose-gel electrophoresis) preclude its use on partially degraded DNA and in high-throughput population studies running smaller fragments on automated DNA analyzers. Here, we propose an improved molecular method based on the amplification of a shorter section of the sdY gene for sex-determination in Atlantic salmon and brown trout. Designed primers were multiplexed with several microsatellites and provided reliable sexing for 97 % of the individuals tested. We successfully applied this method to both fresh and archived tissues from 16 individuals (9 populations) of brown trout and 19 individuals (14 populations) of Atlantic salmon. This molecular marker also includes important length polymorphism providing useful Y-linked phylogeographic information.