Documentation of the biodiversity of eukaryotic algae from desert systems is sparse. Our objective was to characterize microalgae from soil samples collected throughout Joshua Tree National Park, California, USA. Morphological, life-cycle, and DNA sequence data were collected for 100 microalgal isolates distributed over 18 sites in Joshua Tree National Park. Phylogenetic analysis of the 18S rDNA data separated the green algae into 15 major clades—10 in the class Chlorophyceae and 5 in the class Trebouxiophyceae—containing 2 or more lineages plus 9 lineages represented by a single isolate. Five isolates belonging to the class Xanthophyceae and 2 isolates belonging to Eustigmatophyceae were also identified. Some green algal isolates could be placed with confidence in known genera including Bracteacoccus, Chlorosarcinopsis, Myrmecia, Neochlorosarcina, Scenedesmus, and Stichococcus, whereas several green isolates could not be assigned to known genera based on morphological or molecular data. Both morphological and molecular data were important to identifying this biodiversity. Due to the paucity of informative morphological characters, morphology alone does not capture the species diversity found at sites. Molecular data are a richer source of characters with which to identify the algae, but more representative sequences of soil algae are needed in public databases to make identification of any new taxa straightforward. Overall, our data suggest that the biodiversity of these hot deserts still is largely unknown and unexplored.