Streptophytes are one of the major groups of the green lineage (Chloroplastida or Viridiplantae). During one billion years of evolution, streptophytes have radiated into an astounding diversity of uni- and multicellular green algae as well as land plants. Most divergent from land plants is a clade formed by Mesostigmatophyceae, spp. and Chlorokybophyceae. All three lineages are species-poor and the Chlorokybophyceae consist of a single described species, In this study, we used phylogenomic analyses to shed light into the diversity within using a sampling of isolates across its known distribution. We uncovered a consistent deep genetic structure within the isolates, which prompted us to formally extend the Chlorokybophyceae by describing four new species. Gene expression differences among species suggest certain constitutive variability that might influence their response to environmental factors. Failure to account for this diversity can hamper comparative genomic studies aiming to understand the evolution of stress response across streptophytes. Our data highlight that future studies on the evolution of plant form and function can tap into an unknown diversity at key deep branches of the streptophytes.