Brown trouts native to the Ebro river system in the Northeast region of Spain provide an excellent example of seriously threatened populations with immediate risk of extinction. To document the genetic relationship among native and restocked brown trouts confined in this geographical location (Mediterranean drainage), we have analysed mitochondrial DNA (mtDNA) variation after selective amplification and RFLP analysis with 7 restriction enzymes. We primarily focused our study on individuals collected from different tributaries of the Ebro river system. Four distinct mtDNA genotypes were identified: AAAAAAA, ABBBAAA, ABBBBBB and BBBBABB. The AAAAAAA genotype was present at maximum frequency in affluent systems or tributaries under low or no restocking action, and therefore was inferred to be a Mediterranean native brown trout genotype. The ABBBBBB and BBBBABB genotypes were characteristic Atlantic stocks currently employed in the restocking programs. Similarly, the ABBBAAA genotype was found in tributaries under intensive restocking but its origin is unknown. We conclude that the genetic integrity of our Mediterranean brown trout taxon is being compromised through inappropriate hatchery broodstock selection and recommend that genetic data be incorporated into management strategy for native brown trout conservation.