AbstractThe conjugating green algae (Order Zygnematales and Desmidiales) are charophyceans, that is, they are among the green algae most closely related to land plants. Compared to the five other groups of charophyceans (Mesostigma, Klebsormidiales, Chlorokybales, Coleochaetales, Charales), the conjugating greens are by far the most diverse taxonomically (6 families, about 55 genera, 3–4000 species) and very widespread in distribution (including common species such as Spirogyra, Zygnema, Cosmarium, and Staurastrum). Thallus shape ranges from simple, oblong unicells (saccoderm desmids, or Mesotaeniaceae), to unbranched filaments of cells with smooth walls (Zygnemataceae), to elaborately ornate and deeply incised unicells or filaments made of such cells (placoderm desmids, in four families). Prior analyses of sequences of the chloroplast‐encoded gene rbcL (1354 bp, or 95% of the gene) sampled from all six families showed that two of the traditional families (saccoderms and filamentous forms) are paraphyletic, i.e. they do not form two independent clades, although the component genera may together constitute a monophyletic group. In contrast, three families of placoderm desmids formed three monophyletic groups within a larger clade. New analyses using partial sequences from the mitochondrial gene coxIII (608 bp, or 76% of the gene) for 21 species of conjugating green algae from all six families supported the conclusions of the rbcL analysis, although bootstrap support for each gene along was weak for many branches. The coxIII sequences are more variable than rbcL and have proportionately more parsimony‐informative sites (43% vs. 34%, respectively). Combining the two genes improved the resolution of the resulting tree: (1) bootstrap support increased for all branches, particularly in regards relationships of placoderm desmids; and (2) monophyly of a clade containing filamentous and saccoderm desmids was strongly supported. We conclude that phylogeny of conjugating green algae will be much improved by sampling from additional genes.