Phosphate is involved in fatty acid biosynthesis in microalgae cells. The influence of phosphate concentrations on polyunsaturated fatty acids' profile, elongation, and desaturation of fatty acids chains during their biosynthesis still remains unclear. Protein ketoacyl-ACP synthase-I (KAS-1 ) is required for the addition of malonyl-CoA for the elongation of butyryl-ACP from a 4-to 14-carbon chain. The omega-6 (ω-6 FAD ) and omega-3 desaturases (ω-3 FAD ) are involved in the insertion of double bonds into the pre-formed fatty acid chains. This study reports the effect of phosphate concentrations on the growth, fatty acid content, and level of KAS-1, ω-6 FAD, and ω-3 FAD transcripts in Chlorella KS-MA2 strain at the stationary growth phase. Phosphate was tested at concentrations 9, 18, 36, and 72 μM and the fatty acid content was analyzed using gas chromatography. Gene expression levels were measured using quantitative real-time-PCR. Results showed that cell growth, amount of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFAs) did not significantly (P > 0.05) differ among tested treatments. The highest concentration of α-linolenic acid and the expression level of ω-3 FAD were recorded with 72 μM of phosphate in culture medium. The KAS-1 and ω-6 FAD transcripts levels decreased when the concentration of phosphate was below 36 μM. Phosphate in F/2 medium was sufficient to enhance the biosynthesis of major fatty acids in Chlorella sp.