The Eastern Italian Alps (South Tyrol) is a connection area between continental Italy and the northern Alps. Various local factors, such as the heterogeneous environment, complex historical events, and different mobility patterns, may have influenced the genetic makeup of early medieval alpine groups. However, no ancient genetic data from these groups are currently available. This study provides a first picture of the ancient mitochondrial DNA (mtDNA) diversity of alpine groups from four locations in South Tyrol (Adige, Isarco, Venosta, and Merano). In total, 94 ancient mitogenomes of individuals (dated from 400 to 1100AD) were reconstructed by shotgun sequencing and a mtDNA capture approach. Moreover, stable isotope ratios (δ13C, δ15N, δ34S) were analyzed in a subset of 32 individuals. The results indicate different mtDNA haplogroup distributions among the alpine locations and the presence of rare lineages besides a possible maternal relatedness between individuals buried in the same and in diverse archaeological contexts. The study also shows differences in the genetic and mobility patterns (δ34S) between individuals from the central and north-eastern parts (Adige, Merano, Isarco) and those from the north-western part of South Tyrol (Venosta). These results suggest genetic exchanges with allochthonous people in the first group probably linked to high mobility and to geomorphological, historical, and socio-cultural factors. Comparisons extended to present-day alpine populations also suggested maternal genetic continuity in this alpine area. Finally, stable isotope (δ13C, δ15N, δ34S) data provided further support for regional differences in the diet of past alpine groups possibly linked to altitude and/or social status.