Hemoglobin (Hb) extracted from bloodstains was investigated using cation-exchange high-performance liquid chromatography (HPLC). Among the HbA1 fractions, the amount of HbA1d increased linearly as the ages of the bloodstains increased, although the amount of HbA1c remained virtually unchanged. As HbA1 fractions are Hb modified with glucose or glycolytic intermediates, the total Hb were separated into glycated and non-glycated Hb fractions using affinity chromatography to investigate the effects of glucose on the age-induced Hb changes. The amounts of glycated Hb increased in proportion to the blood glucose level, and the increased glycated Hbs were eluted at retention times that corresponded to those of HbA1d and HbA0, although HbA1c peak area was virtually unchanged. The amounts of both glycated and non-glycated HbA1d increased with bloodstain aging, and the proportion of non-glycated HbA1d relative to the total HbA1d was greater than that of glycated HbA1d. These findings show that glucose has little effects on the elevation of HbA1d in bloodstains that occurs as they age. Furthermore, measuring HbA1d levels in bloodstains using HPLC may prove useful for the determination of their ages.