Microalgae are potential feedstocks for the commercial production of carotenoids, however, the metabolic pathways for carotenoid biosynthesis across algal lineage are largely unexplored. This work is the first to provide a comprehensive survey of genes and enzymes associated with the less studied methylerythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway as well as the carotenoid biosynthetic pathway in microalgae through bioinformatics and comparative genomics approach. Candidate genes/enzymes were subsequently analyzed across 22 microalgae species of lineages Chlorophyta, Rhodophyta, Heterokonta, Haptophyta, Cryptophyta, and known Arabidopsis homologs in order to study the evolutional divergence in terms of sequence-structure properties.