In primitive and higher plants, intracellular storage lipid droplets (LDs) of triacylglycerols are stabilized with a surface layer of phospholipids and oleosin. In chlorophytes (green algae), a protein termed major lipid-droplet protein (MLDP) rather than oleosin on LDs was recently reported. We explored whether MLDP was present directly on algal LDs and whether algae had oleosin genes and oleosins. Immunofluorescence microscopy revealed that MLDP in the chlorophyte Chlamydomonas reinhardtii was associated with endoplasmic reticulum subdomains adjacent to but not directly on LDs. In C. reinhardtii, low levels of a transcript encoding an oleosin-like protein (oleolike) in zygotes-tetrads and a transcript encoding oleosin in vegetative cells transferred to an acetate-enriched medium were found in transcriptomes and by reverse transcription-polymerase chain reaction. The C. reinhardtii LD fraction contained minimal proteins with no detectable oleolike or oleosin. Several charophytes (advanced green algae) possessed low levels of transcripts encoding oleosin but not oleolike. In the charophyte Spirogyra grevilleana, levels of oleosin transcripts increased greatly in cells undergoing conjugation for zygote formation, and the LD fraction from these cells contained minimal proteins, two of which were oleosins identified via proteomics. Because the minimal oleolike and oleosins in algae were difficult to detect, we tested their subcellular locations in Physcomitrella patens transformed with the respective algal genes tagged with a Green Fluorescent Protein gene and localized the algal proteins on P. patens LDs. Overall, oleosin genes having weak and cell/development-specific expression were present in green algae. We present a hypothesis for the evolution of oleosins from algae to plants.