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The integration of taxonomic and ecological traits is a more balanced approach in the systematic identification of living organisms. We isolated ten strains of Dunaliella from different geographical regions of Iran, including six terrestrial and four aquatic taxa. All strains belonged to the section Dunaliella based on the morphological and molecular investigation. First, optimal salinity was determined for bio-pigment accumulation. Each alga had its own optimum salinity, and the range of 1.5 to 2.5 M NaCl was further studied. Dunaliella parva YzS produced the maximum biomass density (231.92 mg L−1), while Dunaliella salina MrL accumulated the highest value of total carotenoid (1.72%AFDW). At the early stationary growth phase, the algae had a 11.7 to 41.7% lipid content. FAMEs varied from C10 to C24 with one or more double bonds. The results showed highly significant levels of palmitic (C16) and α-linolenic (C18:3n3) acids. According to the fatty acid profiles, the clustering pattern aligns with the sample origin. Compared to aquatic strains, terrestrials were more resistant to harsh conditions, e.g., dry conditions due to the synthesis of very long chain fatty acids. Lipid compositions may therefore be attributable to the environmental origins of taxa. The fatty acid composition was affected by the environmental conditions, while the accumulation of carotenoid was dependent on the biological and genetic entity of algal taxa. Examination of habitat parameters revealed that aquatic strains were well adapted to maintain homeostasis in higher salinity and conductivity; terrestrial strains were much better able to tolerate high concentrations of nitrate and phosphate.