Solanaceae is one of the prominent plant families that provide medicine and food. For the diagnosis of its species (Physalis peruviana and Physalis angulata), the use of molecular markers with reliable and precise nuclear ribosomal DNA (nrDNA) ITS sequences has helped in the presumption of molecular evolution and phylogenetic studies of the plants. It precisely measured the variance between two samples of Physalis L. Purified fragments' sequencing using BLAST to align each sequence, evaluate its relationship with other sequences, and confirm the species of different sequences. The genetic analysis program, MEGA V.11, aided in performing multiple sequence alignment and phylogenetic analysis. The first sample showed a relation to the species Physalis peruviana voucher Smith 217 (WIS) placed in the GenBank with the serial number DQ314161.1 by 96.91%, which belonged to a strain located in the USA. The researcher registered the species as Physalis peruviana isolate AH-ZE1, with serial number OQ616506.1. However, the second sample, after contrasting it with the global species sequences, indicated a connection to the species Physalis angulata isolate LHR28I deposited in the Genome Bank with the serial number MK412130.1 at a rate of 98.02%, which belonged to a Spanish strain. The species' registration by the researcher continued as Physalis angulata isolate AH-ZE1, sequence number OQ616509.1. The current study results revealed the diagnosis of two species of Physalis identified for the first time in Iraq, as they incur registration in the National Center for Biotechnology Information. The recording depended on the molecular characteristics' differences in the ribosomal DNA (rDNA) region, knowing the evolutionary relationship between the Physalis species and comparing them to the sequences found in the GenBank for previously defined types. The results also showed that the ITS2 region provided success ability. Therefore, using other DNA barcodes as auxiliary factors to distinguish between Physalis species would be beneficial.