Despite extensive knowledge of mitochondrial DNA (mtDNA) variation in European brown trout (Salmo trutta) populations, little is known about their nucleotide sequence variation in North America. The objective of this study was to quantify single nucleotide polymorphisms (SNPs) at the ND-1 mtDNA locus of 62 brown trout from hatcheries in Michigan and Wisconsin as well as Michigan streams and Lake Michigan. We identified 25 SNPs that characterized nine distinct mtDNA haplotypes in the Wild Rose, Gilchrist and Seeforellen brown trout strains. Although most SNPs were represented by synonymous nucleotide substitutions, three individuals of the Seeforellen strain had non-synonymous nucleotide changes. MtDNA haplotypes identified in North American brown trout in this study showed nucleotide similarity at the ND-1 locus to brown trout from northern Europe.