Conservation and management of wild species needs a detailed understanding of their population structure and dynamics. For instance, there are increasing evidence for sex-specific life history trajectories, even in species with limited morphological sexual dimorphism. In these species, identifying the sex of individuals in some or all life stages is a methodological challenge, which requires the development of new tools. Building on recent findings in molecular biology, we developed a new protocol for sex identification in Atlantic salmon (Salmo salar), aiming at the following criteria: accurate sex identification, fast diagnostic, applicable to large sample size, applicable to degraded DNA extracted from e.g. fish scales, and safe for the operator by limiting health and safety hazard. The protocol relies on multiplex Real-Time PCR using a set of specific primers. We tested its accuracy on a test set of 90 DNA samples from individuals of known morphological sex, and demonstrated its applicability on a large DNA sample from 384 individuals of unknown sex.