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Introduction: A three-factors self-reporting questionnaire, consisting of 25 items, related to behavior/negative affect, affect/behavioral aggression, and negative cognition towards homosexual people, was administered to a sample of 200 farmer workers recruited in various ways from two Bryansk and Kursk  regions of  Russian Federation. Aim: The objective of this paper is to validate the HS in the Russian sample in Russian language. Methods: A Russian translation of the HS was carried out by two bilingual translators, after which an English native translated the Russian test back into the English language. Main Outcome Measures: Together with the translated version of HS was administrated a biodata questionnaire including the main information as age, level of education, partnership status, faith and sex orientation. Results : Analyzing obtained data of 200 subjects (100 (50%) female and 100 (50%) male) with ages ranging from 18 to 50 years (mean age = 21.19±2.65; SD: 8.82) showed an overall Cronbach\'s α coefficient 0.92 of the internal consistency. All three domains had a good alpha coefficient (three higher than .91), whereas in the total score was .87. The test-retest reliability results: behavior/negative affect was r = 0.79 (P < 0.0001), affect/behavioral aggression was r = 0.82 (P < 0.0001), negative cognition was r = 0.74 (P < 0.0001) and the HS total score was r = 0.93 (P < 0.0001). Conclusions: This study offers a new tool to assess homophobia in Russian population. The Russian validation of the HS in Russian language revealed the use of this self-report test to have good psychometric properties. Therefore, this scale is a necessary diagnostic instrument to detect subtle homophobic attitudes toward homosexual people and useful in the preventive programs of homophobic behavior, in the clinical praxis.