The large-scale migration of Afghans following four decades of war and insecurity has presented them with numerous challenges in their host countries. One of these challenges is acculturation stress, which can affect the mental and physical health of immigrants. The aim of the study was to examine the validity and reliability of the acculturation stress questionnaire among Afghan immigrants living in Iran. The statistical population included all Afghans over 18 years of age living in Iran, from whom 510 people were selected using convenience sampling (252 people for exploratory and confirmatory analysis of the seven-factor model and 258 people for confirmatory analysis of the four-factor model). Data were collected through an online link and a printed version at the Afghan embassy. Confirmatory factor analysis showed that the seven-factor model is not confirmed; However, exploratory factor analysis extracted a four-factor structure including “perception of discrimination”, “longing for homeland/compatriot”, “distress and insecurity”, and “feeling of alienation”, which explained a total of 57.042% of the variance. The reliability of the subscales was between 0.80 and 0.92 and the internal consistency of the entire scale was 0.95. The findings indicate that this tool can be useful in identifying cultural stresses of Afghan immigrants and designing supportive interventions.