Red rice is the main weed in rice paddy fields. Imidazolinone herbicides in resistant rice cultivars currently provide a unique opportunity to control red rice in large-scale rice fields. However, the continuous use of this technology has resulted in imidazolinone-resistant red rice biotypes. This study aimed to identify the mechanism of herbicide resistance and the frequency and spatial distribution of the known imidazolinone herbicide-resistant alleles in red rice. The nucleotide sequence of the ALS gene indicated that the G₆₅₄E, S₆₅₃D and A₁₂₂T mutations are present in the imidazolinone herbicide-resistant rice cultivars IRGA 422 CL, SATOR CL and PUITÁ INTA CL, respectively. This information and the nucleotide sequence surrounding these mutations were used for the development of single nucleotide polymorphism (SNP) molecular markers to identify the possible mutations that confer herbicide resistance in red rice. This analysis was carried out in a total of 481 plants from 38 populations collected as individuals that escaped control with the herbicides imazethapyr and imazapic in rice paddy fields in Southern Brazil. The G₆₅₄E mutation was the most frequent, being found in 100% and 90.9% of the populations in the 2006/2007 and 20007/2008 seasons, respectively. In addition, the S₆₅₃D and A₁₂₂T mutations were also present either alone or as double or triple mutations in some plants. Target site insensitivity is the predominant mechanism of resistance in red rice resistant to imidazolinone herbicides in Southern Brazil. The high frequency of the S₆₅₃D mutation, the same mutation responsible for the resistance in the rice cultivar largely used in Southern Brazil, indicates that gene flow is occurring from the rice cultivar to red rice. Management practices related to increasing crop sanitation and decreasing of herbicide selection pressure through crop rotation should be enforced to prevent the evolution of herbicide resistance in red rice.