Mitochondrial DNA from Holstein maternal lineages implicated to express cytoplasmic genetic effects on lactation traits was subcloned and screened for molecular polymorphisms. Sixteen of 35 lineages sampled differed from the most common mitochondrial DNA form by at least one restriction endonuclease cleavage site in the 4.3 kilobase segment examined. Variation existed in the region that regulates DNA replication and transcription as well as in transfer and ribosomal ribonucleic acid coding regions of the DNA. The index of nucleotide diversity calculated from polymorphism frequencies indicated that the minimum extent of variation between two random lineages was 1.16 x 10(-4) nucleotide differences per base pair in the segment examined. Presence of a HpaII marker near nucleotide 360 was associated with lower (P less than .001) milk fat percentages. Molecular markers indicated that pedigrees may not be sufficient to separate true cytoplasmic lineages for quantitative genetic analyses. These findings provide a molecular genetic basis for further study of cytoplasmic effects on phenotypic variation in Holstein cattle.