Upland cotton, Gossypium hirsutum L., is highly susceptible to infection by reniform nematode (Rotylenchulus reniformis), which can cause over 10% reduction of cotton yields in Alabama. Detection of reniform nematode (RN) and analysis for molecular variation within its population is important for understanding its interactions with cotton and other host plant species. Restriction analysis of PCR products of ITS1 regions was achieved using four restriction enzymes, HaeIII, HhaI, MspI, and RsaI. These showed similar banding patterns for both male and female populations. However, MspI digestion of ITS1 amplification products showed variants within the combined sex and location effects primarily attributed to a 500 bp fragment that was absent in other restriction digestions. Intra-nematodal variations in 18S and ITS1 rDNA were studied in detail by sequencing a minimum of ten clones in each individual male and female RN isolates in both directions. Multiple sequence alignment of the 18S rDNA sequences showed two major types of sequences within this gene for both male and female RN clones, which could be distinguished at 27 specific sites. Two distinct ITS1 fragments of lengths (550 bp and 720 bp) were observed; referred to as ITS1S and ITS1L respectively. Neighbor-joining analysis was used in revealing the relationships and grouping characteristics between male and female RN clones, with clones grouping together irrespective of sex and isolate. Sequencing of one-third of the 18S and ITS1 rDNA regions provided clear evidence of intra-and inter-nematode variability, in addition to gene conversion events in the 18S rDNA of individual male and female RN clones.