Although the brain is an important part of a person's sexual life, little is known about the correlations between sexual response and brain activation. This study examines brain responses through clinical testing to reveal several brain structures whose activation is relevant to sexual arousal besides psychological testing according to the common traditional values and standards of Iranian society. Based on an initial self-report, 25 homosexual and 25 heterosexual males participated in this test to elucidate the identity and sexual trends via the Minnesota Multiphasic Personality Inventory (MMPI-2). Also, an fMRI technique—by presenting distinct homo- and heterosexual sex erotic pictures during the test—was employed in neural correlates of sexual arousal via blood oxygen level dependent (BOLD) signal measuring and Statistical Parametric Mapping analysis. In five selected homosexuals by the most appropriate MMPI test score, brain activities were significantly detected in the midbrain, amygdala, anterior cingulate gyrus, frontal cortex, orbitofrontal cortex, globus pallidus, thalamus, and putamen when the participant saw the erotic pictures during the test, which were responsive to sexual arousal. Also, bilateral caudate nucleus, left angular gyrus, and bilateral pallidum were activated but five selected heterosexuals showed no activation in these areas (p < 0.001). Likewise, the MMPI method confirmed that homosexuality was relevant to diverse life positions, and biological and socioeconomic aspects. The maximum positive correlations were pairwise found in the parietal lobule by r = 0.61 (p < 0.05) and in the frontal gyrus by r = 0.64 (p = 0.10), and the minimum negative correlations were in globus pallidus by r = − 0.18 (p < 0.10) and r = − 0.16 (p < 0.10) for homosexuals and heterosexuals, correspondingly. These results may be useful for understanding the different neural mechanisms of personality recognition and sexual orientation changes while incorporating the individual's history and experience simultaneously into the assessment.