High-mobility group box 1 protein (HMGB1) is a nuclear DNA-binding protein, which also functions as a pleiotropic cytokine, implicated in the pathology of several different immune-mediated diseases. The purpose of this study was to examine the HMGB1 gene for putative polymorphisms in 103 healthy Caucasian Danish blood donors. A total of six polymorphisms and four mutations were identified in the HMGB1 gene. Subsequent MatInspector estimation revealed that several polymorphisms might have a potential regulatory impact on HMGB1 transcription. This study has characterized genetic variations in the HMGB1 gene locus, which may have a regulating role in the expression of HMGB1, providing the basis for molecular investigations of the HMGB1 gene in different disease settings.