Brown trout Salmo trutta wild stocks sustain a remarkable angling and hatchery-reared fish stocking pressure in waters of Serbia, where four drainage-specific and indigenous mitochondrial DNA haplotypes were reported for the drainage area of the Danube River basin. One of these mitochondrial DNA haplotypes, Da23b, was exclusive for brown trout in headwaters of the Crni Timok River (Grand Timok River system, Eastern Serbia). After its discovery in 2003, brown trout stocking was completely halted and abandoned, and a strict Catch-and-Release fishing regime was issued. However, failure of this regime due to enforcement resulted in decline of brown trout fishery. On checking the aboriginality of brood fish sampled in three headwater forks for artificial brown trout propagation, a novel, drainage-specific mtDNA haplotype was recorded in 54% of brown trout. It was assigned Da23c, due to its similarity (substitutions at the two variable sites) to the hitherto known haplotypes of Da23 group. Its finding and absence of Da23b haplotype imply an oversight in earlier haplotype determination. Incorporation of the non-indigenous haplotypes Da2 and Da-s1, introduced in the area by stocking before 2003, into the gene pool of native brown trout and declining of the latter's stock imposes the need for the more stringently enforced conservational activities (genotyping of each brood fish involved in artificial propagation) and improved fisheries management (Catch-and-Release) in the area.