Squash (Cucurbita moschata D.) is an economically important vegetable of the Cucurbitaceae family. The genetic purity of commercial hybrid seed is crucial for the success of hybrid seed production. The molecular markers like single nucleotide polymorphism (SNP) can efficiently and cost-effectively distinguish the genetic differences among F1 hybrid cultivars. Therefore, in this study, we used 'Fluidigm SNP Genotyping' assay using 27 SNPs to distinguish and purity analysis of registered commercial F1 hybrid cultivars and F1 breeding lines of squash. Of these, eight SNP markers, including CMo-A01, CMo-A02, CMo-A04, CMo-A05, CMo-A12, CMo-A16, CMo-A20 and CMo-A25 can successfully identified heterozygotes from the registered commercial F1 hybrid squash cultivars with 100% accuracy and partial contamination was detected for F1 hybrid squash breeding lines which resulted due to outcrossing. Moreover, the HRM analysis of a registered commercial F1 hybrid cultivar 'Parangsae' with CMo-A03 SNP marker showed 96.30-100% purity of the cultivar. Our results suggest that the 'Fluidigm SNP Genotyping' technology could be a rapid and cost-effective method for cultivar differentiation and genetic purity analysis of F1 hybrids and squash cultivars.