The green algae belonging to the Chlorophyta-the lineage sister to that comprising the land plants and their charophycean green algal relatives (Streptophyta)-have been subdivided into four classes (Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae). Yet the Pedinomonadales, an assemblage consisting of tiny, naked uniflagellates with a second basal body, has no clear affiliation with these classes and the branching order of the crown chlorophytes remains unknown. To gain an insight into the phylogenetic position of the Pedinomonadales and the relationships among the recognized chlorophyte classes, we have sequenced the chloroplast genomes of Pedinomonas minor (Pedinomonadales) and of two trebouxiophyceans belonging to the Chlorellales, Parachlorella kessleri (Chlorellaceae) and Oocystis solitaria (Oocystaceae), and compared these genomes with those of previously examined streptophytes and chlorophytes, including Chlorella vulgaris (Chlorellaceae). Unlike their Chlorella homolog, the three newly investigated chloroplast DNAs (cpDNAs) carry a large rRNA-encoding inverted repeat (IR) that divides the genome into large and small single-copy regions. In contrast to the situation found for ulvophycean and chlorophycean cpDNAs, the gene contents of the IR and single-copy regions are strikingly similar to that inferred for the common ancestor of chlorophytes and streptophytes. The intronless 98,340-bp Pedinomonas genome is among the chlorophyte cpDNAs featuring the smallest size and most ancestral gene organization. All 105 conserved genes encoded by this genome are included in the gene repertoires of Oocystis (111 genes) and Chlorella (113 genes), with just trnR(ccg) missing from Parachlorella cpDNA. Trees inferred from 71 cpDNA-encoded genes/proteins of 16 chlorophytes and nine streptophytes showed that Pedinomonas is nested in the Chlorellales, a group of algae lacking flagella. This phylogenetic conclusion is independently supported by uniquely shared gene linkages. We hypothesize that chlorellalean and pedinomonadalean green algae are reduced forms of a distant biflagellate ancestor that might have also given rise to the other known trebouxiophycean lineages. Our structural cpDNA data suggest that the Chlorellales and Pedinomonadales represent a deep branch of core chlorophytes, strengthening the notion that the Trebouxiophyceae emerged before the Ulvophyceae and Chlorophyceae. Our results further emphasize the importance of secondary reduction at both the cellular and genome levels during chlorophyte evolution.