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The continued search and urgent need for renewable fuel sources have necessitated the exploration of microalgae to identify relevant species for making biofuels. The aim of the study was bioprospecting and screening native microalgae strains from freshwater habitats of the Almaty region, Kazakhstan, to assess the potential for producing biofuel. The studied strains demonstrated simultaneous biomass productivity, lipid productivity, suitable fatty acid composition, and biodiesel properties. The sequence analysis of the ribosomal DNA internal transcribed spacer partial region and ribulose-bisphosphate carboxylase gene (rbcL) led to the identification of five microalgae: Monoraphidium griffithii ZBD-01, Nephrochlamys subsolitaria ZBD-02, Ankistrodesmus falcatus ZBD-03, Parachlorella kessleri ZBD-04, and Desmodesmus pannonicus ZBD-05. P. kessleri had the highest biomass production (1.42 ± 0.08 g L−1 day−1), lipid productivity (29 ± 1.2 g L−1day−1), and C 16 –C 18 fatty acid contents (90%), followed by A. falcatus and M. griffithi. Gas chromatography/mass spectrometry analysis indicated that the dominant fatty acids in these strains were palmitic, stearic, and oleic acids. The calculated biodiesel properties of P. kessleri and A. falcatus based on fatty acid methyl esters (FAME) profiles showed relatively good fuel properties (cetane numbers - 48 and 50; iodine and saponification values - 83.4 and 103.6 g I₂/100 g oil, 260.8 and 199.5 mg KOH g−1), which correlate well with. Our results suggest that P. kessleri and A. falcatus are promising strains for biodiesel production due to their high lipid productivity, fatty acid profile with relatively high content of oleic acid, and suitable biodiesel properties. The isolated native species of microalgae from natural freshwater bodies of the Almaty region present opportunities for further exploitation for the sustainable production of biomass and biodiesel. [Display omitted] · New native strains of green microalgae were isolated and identified. · Fatty acids composition of microalgae strains was investigated. · Microalgae strains found enriched with saturated fatty acids (C16:0 and C18:0). · These strains are the best candidate for application in biofuel. [ABSTRACT FROM AUTHOR]