The morphological characteristics of diatoms are useful for studying their taxonomy. However, the distinction between closely related taxa can be very difficult, especially when the morphological characters are modified by environmental constraints. In the present study, 13 fresh water diatoms were identified morphologically and cultured to have their axenic cultures. To check this PCR primers specific for multilocus genes were designed to amplify and screen 13 fresh water diatom monocultures. Among the multilocus PCR primers (DRR3, scfcpA, Lhcf11, SIT1, SIT3, SIT4, LOC101218388, COI-5P, rbcL, rbcL-3P, LSU D2/D3, UPA, psaA and 18S rRNA) we found that psaA gene, a plant pigment chlorophyll-based primer amplified all the diatoms. Out of 13 diatom amplicons only two fresh water diatoms DNA were sequenced. This included Cyclotella meneghiniana due and Sellaphora pupula. The Sanger sequencing results thus established that morphologically identified diatom, Sellaphora pupula exhibited close phylogeny to Sellaphora whereas Cyclotella meneghiniana had close lineage to marine diatom Thallosiosira.