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Background Although the number of new HIV infections is declining in most regions of the world, the Middle East is one of the regions with a rapidly growing HIV epidemic, with Egypt having the fastest-growing epidemic, with a 76 percent increase in the number of cases. One of the major factors contributing to this trend is the general public’s lack of knowledge about the disease. The HIV Knowledge Questionnaire-18 (HIV-KQ-18) is one of the most widely used instruments for assessing HIV/AIDS knowledge and has been translated into several languages. This study examined the validity of the Arabic version of the HIV-18-KQ as well as its adaptation among Arab undergraduates. Methods The HIV-18-KQ was both forward and back-translated. The translation was reviewed by an expert committee of eight experts. The final version was created and distributed to undergraduates from five Arabic countries: Egypt, Sudan, Yemen, Jordan, and Algeria. The validity of the Arabic version of the HIV-18-KQ was evaluated using internal consistency and construct validity. Internal consistency was tested using the Kuder-Richardson formula 20 (KR-20), and construct validity was evaluated using an exploratory factor analysis with a polychoric correlation matrix. Results The majority of the translated items were easy to understand. The Arabic HIV-18-KQ was deemed culturally appropriate by the expert committee. This study included 1745 university students, including 956 (54.5%) males and 798 (45.5%) females, with 33.4% from Egypt. Based on the acceleration factor approach to interpreting the scree plot in the factor analysis, it was preferable to use only one factor, which is consistent with the original version of HIV-45-KQ. The KR-20 value was 0.73, indicating good internal reliability. Conclusion This study demonstrates that the Arabic version of the HIV-18-KQ is a valid and reliable tool for assessing HIV-related knowledge in Arabic-speaking countries.