The practice of monoculture and the use of one-mode-action herbicides for more than 10-year period can cause development of resistant jungle rice [(Echinochloa colona (L.)] to photosystem II inhibitors. Jungle rice is one of the most troublesome weeds in the sugarcane fields of Iran. Laboratory and greenhouse testing was carried out from 2013 to 2016 to investigate the development and determine the origin of metribuzin-resistant jungle rice biotypes in the sugarcane fields of Khuzestan province. Five populations of jungle rice (1 susceptible and 4 suspected resistant) were collected and assayed. The suspected populations were first screened using discriminating concentrations of mertibuzin. To determine the resistance factor for R-biotypes, bioassay trials were conducted in a greenhouse. Then, PCR sequencing markers used in the laboratory and PCR products were subjected to pair-end sequencing by Eurofins Scientific to determine the molecular base of resistance in the R-biotypes. The results of seedling bioassays showed that four biotypes were resistant to metribuzin. The weed control efficiency of metribuzin for the R4, R2, R3 and R1 biotypes were 12.38%, 27.24%, 59.29% and 75.21%, respectively. The resistance factor for the R4 and R2 biotypes were 58.51 and 30.23 respectively; ranking these as resistant biotypes. Gene sequencing studies found an effective mutation at position 232 of the psbA gene that resulted in amino acid substitution from Serine to Glycine at residue 264 in the D1 protein in R-biotypes versus S-biotypes. These results show that target site resistance to metribuzin is rapidly developing in sugarcane fields in northern Khuzestan province. This could stem from the spread of resistant biotype seeds through irrigation water from the Karun Agro-industry fields (first reported sites of resistance) or by herbicide selection pressure.