<p>Six carotenogenic strains of halophilic microalga <i>Dunaliella</i> were isolated for the first time from hypersaline environments in Crimea: Lakes Sasyk-Sivash, Sakskoye, Oyburskoye, Moynakskoye, Genicheskoye, and Sivash Bay and characterized through an integrative approach combining morphological, molecular, and biochemical analyses. Unialgal cultures were obtained and examined by light microscopy, scanning electron microscopy, and imaging flow cytometry (FlowCam). Morphometric analysis revealed two distinct size classes: strains from Lakes Sakskoye and Genicheskoye possessed significantly smaller cells than those from the remaining four localities. Discriminant analysis of FlowCam data demonstrated that combining morphometric and chromaticity parameters yielded optimal classification accuracy, substantially exceeding that achieved by either parameter set alone. Phylogenetic reconstruction based on the nuclear ITS region resolved the isolates into two species: <i>Dunaliella salina</i> (Lakes Sasyk-Sivash, Oyburskoye, Moynakskoye, and Sivash Bay) and <i>Dunaliella parva</i> (Lakes Sakskoye and Genicheskoye), revealing substantial genetic diversity among geographically proximate hypersaline habitats. Combined stress treatment (high irradiance and exogenous H₂O₂) induced carotenoid hyperaccumulation in all strains. Spectrophotometric and HPLC analyses showed that <i>D. salina</i> strains accumulated markedly higher total carotenoid levels, with β-carotene as the dominant pigment, whereas <i>D. parva</i> strains exhibited approximately ten-fold lower carotenoid content, dominated by lutein. Among the <i>D. salina</i> isolates, the Sivash Bay strain displayed the highest carotenoid productivity. These findings establish the Crimean <i>Dunaliella</i> isolates as promising natural sources of β-carotene and lutein for biotechnological applications and highlight the value of polyphasic taxonomy for strain selection.</p>