Double-stranded DNA breaks induce serine phosphorylation of histone H2A.X, producing γ-H2A.X foci that are then recognized by DNA damage response pathway proteins. Formation of γ-H2A.X is therefore critical for the repair of DNA double-stranded breaks and maintenance of genomic stability, and defects in the recognition or repair of double-stranded breaks can result in tumorigenesis. However, key details regarding the formation of γ-H2A.X and its possible role in tumorigenesis remain elusive. Here, we report a previously unknown phosphorylation site on H2A.X, Tyr39. Phosphorylation at this site is induced by ionizing radiation and is a prerequisite for γ-H2A.X formation. Increased phosphorylation of H2A.X at Tyr39 was observed in multiple cancer cell lines, and we found that H2AX Tyr39 phosphorylation positively correlated with histological grade, tumor size and tumor node metastasis stage, and negatively correlated with survival. We also identified a potential role for eyes absent 2 (EYA2) in regulating H2A.X Tyr39 phosphorylation. Our study supports an important role for H2AX Tyr39 phosphorylation in γ-H2A.X formation and cancer progression.