Asian cultivated rice (Oryza sativa L.), an important cereal crop worldwide, was domesticated from its wild ancestor 8000 years ago. During its long-term cultivation and evolution under diverse agroecological conditions, Asian cultivated rice has differentiated into indica and japonica subspecies. An effective method is required to identify rice germplasm for its indica and japonica features, which is essential in rice genetic improvements. We developed a protocol that combined DNA extraction from a single rice seed and the insertion/deletion (InDel) molecular fingerprint to determine the indica and japonica features of rice germplasm. We analyzed a set of rice germplasm, including 166 Asian rice varieties, two African rice varieties, 30 accessions of wild rice species, and 42 weedy rice accessions, using the single-seeded InDel fingerprints (SSIF). The results show that the SSIF method can efficiently determine the indica and japonica features of the rice germplasm. Further analyses revealed significant indica and japonica differentiation in most Asian rice varieties and weedy rice accessions. In contrast, African rice varieties and nearly all the wild rice accessions did not exhibit such differentiation. The pattern of cultivated and wild rice samples illustrated by the SSIF supports our previous hypothesis that indica and japonica differentiation occurred after rice domestication under different agroecological conditions. In addition, the divergent pattern of rice cultivars and weedy rice accessions suggests the possibility of an endoferal origin (from crop) of the weedy rice included in the present study.