Intraspecific studies of red algae have relied on nuclear or plastid markers rather than mitochondrial data to address questions of systematics, biogeography or population genetics. In this study, primers were developed that spanned the noncoding intergenic region between the mitochondrial cytochrome oxidase subunit 2 and cytochrome oxidase subunit 3 genes. These primers were demonstrated to be successful on a variety of red algae in different orders: Gracilariales, Bonnemaisoniales and Ceramiales (families: Delesseriaceae, Ceramiaceae and Rhodomelaceae). Amplification products were between 450 and 320 bp in length, with variation in length shown among geographically distant isolates within a species. The region was variable within a single species, as shown for Bostrychia moritziana and B. radicans, and within populations of Caloglossa leprieurii. In the latter species, four mitochondrial haplotypes were observed in isolates from a single locality in Woolooware Bay, New South Wales, Australia. Analysis of hybrids between different mitochondrial haplotypes of B. moritziana revealed that the mitochondria are maternally inherited in this species. This is the first report of a mitochondrial marker that is variable within red algal populations and may lead to a better understanding of the population ecology of these important marine organisms.