This study aimed at exploring microalgal heterogeneity from fresh water samples collected from inland water bodies in the heavily built city of Singapore. Culturable pure isolates (n = 94) were subject to an in-house microalgal DNA extraction method and LSU rDNA sequencing. Isolates were analysed for their predominance and distribution. A total of 17 different algal genera were identified (H = 2.8, EH = 0.6), of which Scenedesmus spp. and Chlorella spp. constituted 27.5% and 21.3% of isolates respectively, followed by Micractinium spp. (18.8%) and Chlamydomonas spp. (12.5%). We also report 16 new microalgal strains from this region. The data is important from an ecological and biotechnological perspective.