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Fixing the genomic composition and multiplication through true potato seed (TPS) is an important challenge in autotetraploid potato. Disrupted meiotic cDNA (DMC1) is a meiotic gene that plays a central role in DNA recombination through crossing over in meiosis. Using the <i>Arabidopsis</i> DMC1 (<i>AtDMC1</i>) gene sequence, we retrieved <i>Solanum tuberosum</i> DMC1(<i>StDMC1</i>) from the diploid potato genome, and subsequently, sense and antisense regions of the <i>StDMC1</i> gene were amplified in potato cv. Kufri Jyoti. The sense and antisense fragments were confirmed by Sanger-sequencing and cloned in the pRI101 vector. <i>Agrobacterium</i>-mediated transformation of the RNAi construct resulted in 44% transformation efficiency, and a total of 137 mutant lines were obtained. These mutant lines were further validated through pollen viability testing, and selected lines were used for gene expression analysis. The acetocarmine-based pollen staining showed reduced pollen viability ranging from 14 to 21% in four DMC1 mutant lines (DMC4-37, DMC4-41, DMC6-20, and DMC6-21), as compared to the Kufri Jyoti control plants, which on average exhibited 78% pollen viability. The phenotypic data was supported by the reduced expression of the <i>StDMC1</i> gene in these four mutant lines compared to the control Kufri Jyoti. The results confirmed the generation of <i>StDMC1</i> knockdown lines. This is the first report of <i>StDMC1</i> mutant line generation in tetraploid potatoes and will be a step forward in generating non-recombinant mutants through sexual reproduction in potatoes.