Introduction: The aim of this study was to determine the knowledge, attitude, and practice of AIDS patients with regard to the prevention of disease transmission in Yazd and Ahvaz, Iran. Methods: In this cross-sectional study, we evaluated the knowledge, attitude, and practice of 130 patients, selected via simple sampling, with regard to the prevention of disease transmission in Yazd and Ahvaz, Iran (65 cases from each city). Data were collected by researchers, using a questionnaire and a checklist. For statistical analysis, descriptive and analytical tests were performed, using SPSS version 16. Results: In this study, the sample included 130 AIDS patients from Yazd and Ahvaz. Overall, 67.6% of the patients were male; on the other hand, 29.2% and 32.3% of the participants from Yazd and Ahvaz were female, respectively. As the findings revealed, 44.6% and 55.4% of the patients from Yazd and Ahvaz were married, respectively. The mean scores of knowledge, attitude, and practice of the patients regarding the prevention of HIV transmission were 40.31 ± 3.3, 41.92 ± 8.8, and 43.28 ± 5.3 in Yazd, respectively. Also, the mean scores of knowledge, attitude, and practice with regard to the prevention of HIV transmission were 40.68 ± 3.2.5, 42.52 ± 9.05, and 43.24 ± 6.08 among patients from Ahvaz, respectively. Overall, the results showed that the knowledge, attitude, and practice of most patients were at a medium level. Also, most of the participants from Yazd (70.8%) and Ahvaz (56.9%) had obtained information at AIDS counseling centers. Conclusions:The results highlighted the need to educate patients regarding the prevention of HIV transmission.