DNA was extracted from the pumpkin seeds. Five microsatellite markers from plant chloroplast were used for genetic analysis. Except ccSSR-4 could not be amplified by PCR, the rest five microsatellite markers were yielded single PCR bands. The DNA sequences for the five microsatellite markers were analyzed using BLAST. The results showed that the pumpkin seeds belong to Cucurbita pepo subsp. fraternal TCN#1158 or TCN#1156.