Mycosporine-like amino acids (MAAs) are widespread in various microbes and protect them against harsh environments. Here, four different Aphanizomenon species were isolated from severely eutrophic waterbodies, Lake Dianchi and the Guanqiao fishpond. Morphological characters and molecular phylogenetic analysis verified that the CHAB5919, 5921, and 5926 strains belonged to the Aphanizomenon flos-aquae clade while Guanqiao01 belonged to the Aphanizomenon gracile clade. Full wavelength scanning proved that there was obvious maximal absorption at 334 nm through purified methanol extraction, and these substances were further analyzed by HPLC and UPLC-MS-MS. The results showed that two kinds of MAAs were discovered in the cultured Aphanizomenon strains. One molecular weight was 333.28 and the other was 347.25, and the daughter fragment patterns were in accordance with the previously articles reported shinorine and porphyra-334 ion characters. The concentration of the MAAs was calibrated from semi-prepared MAAs standards from dry cells of Microcystis aeruginosa PCC7806 algal powder, and the purity of shinorine and porphyra-334 were 90.2% and 85.4%, respectively. The average concentrations of shinorine and porphyra-334 were 0.307-0.385 mu g/mg and 0.111-0.136 mu g/mg in Aphanizomenon flos-aquae species, respectively. And there was only one kind of MAAs (shinorine) in Aphanizomenon gracile species.,with a content of 0.003-0.049 mu g/mg dry weight among all Aphanizomenon gracile strains. The shinorine concentration in Aphanizomenon flos-aquae was higher than that in Aphanizomenon gracile strains. The total MAAs production can be ranked as Aphanizomenon flos-aquae > Aphanizomenon gracile.