Internal transcribed spacers (ITS1 and ITS2) and the intergenic spacer (IGS) of the nuclear ribosomal RNA region were used to examine the genetic diversity in boll weevils. Weevils were obtained from three locations in Texas, one location in northeastern Mexico, and three locations in southern Arizona. The Texas and Mexico samples originated from cultivated cotton while the Arizona collections were obtained from thurberia cotton, a wild relative of cultivated cotton. The boll weevil IGS is large and contains 5 histone gene sequences. We found restriction pattern differences in a portion of the IGS that differentiated between the Arizona populations from thurberia cotton and weevil populations in Texas and Mexico. Nucleotide variations in the ITS2 revealed one and two base pair differences that are indicative of three distinct weevil populations corresponding to the Texas, Mexican, and Arizonan populations. ITS1 lacked diagnostic polymorphism. The results support previous mtDNA research indicating that the Arizona “thurberia” weevils are a separate population that is isolated from the cotton pest weevils. The ITS2 results combined with some previously observed rare mtDNA haplotypes provide the best evidence so far that the Mexican boll weevil is a distinct genetic entity.