Maternal lineage effects, probably indicative of mitochondrial DNA (mtDNA) differences, may be important for milk production and reproductive success in dairy cattle (Bos taurus). Sequence variation of mtDNA in 36 maternal lineages of dairy cattle was studied with animal models to assess effects on milk production and reproductive traits. Cattle within maternal lineages defined by registered pedigrees were assumed to be uniform for the nucleotide sequences examined. Sequence polymorphisms of bovine mtDNA were shown to be associated with milk production, reproduction, and health costs incurred. One particular base-pair substitution was associated with additional production of 842 kg milk and 37 kg milk fat per cow per lactation. Another single base-pair substitution was associated with a decrease of 36 days and one unsuccessful breeding between successive calvings. Effects of this size are economically important and have broad implications in genetic selection of dairy cattle.