Loss of pancreatic islet β-cell mass and β-cell dysfunction are central in the development of type 2 diabetes (T2DM). We recently showed that mature human insulin-containing β-cells can convert into glucagon-containing α-cells ex vivo. This loss of β-cell identity was characterized by the presence of β-cell transcription factors (Nkx6.1, Pdx1) in glucagon(+) cells. Here, we investigated whether loss of β-cell identity also occurs in vivo and whether it is related to the presence of (pre)diabetes in humans and non-human primates. We observed an 8x increased frequency of insulin(+) cells co-expressing glucagon in diabetic donors. Up to 5% of the cells that were Nkx6.1(+) but insulin(-) co-expressed glucagon, which represents a 5x increased frequency compared to the control group. This increase in bihormonal and Nkx6.1(+)glucagon(+)insulin(-) cells was also found in islets of diabetic macaques. The higher proportion of bihormonal cells and Nkx6.1(+)glucagon(+)insulin(-) cells in diabetic macaques and humans was correlated with the presence and extent of islet amyloidosis. These data indicate that loss of β-cell identity occurs in T2DM and could contribute to the decrease of functional β-cell mass. Maintenance of β-cell identity is a potential novel strategy to preserve β-cell function in diabetes.