This study focuses on polychrome glass tesserae from the palaeo-Christian mosaic, still in situ, which decorates the vaults of the votive chapel of St. Maria Mater Domini in Vicenza (Veneto region, NE Italy). Aims are characterisation of the “base composition” of the glassy matrix and identification of opacifiers and colourants and these are performed by means of a multi-methodological approach (SEM-EDS, EMPA, XRPD, IS). Systematic comparisons between the Vicenza tesserae and those from the votive chapel of St. Prosdocimus in Padova, part of the remains of the only other palaeo-Christian mosaic located in the Veneto, coeval in age and with comparable colour palettes, are also carried out in order to improve our knowledge of production technologies of these materials during the 6th century AD in the Northern Adriatic area. Textural, chemical, diffractometric and spectroscopic data indicate multiple production technologies in the Vicenza tesserae, with evidence of several glassy matrices, opacifiers/pigments and/or ionic colourants/decolourants, all variously mixed in order to obtain the desired shades. In particular, the matrices of the Vicenza tesserae, mostly comparable with compositional reference groups typical of both Roman and Late Roman times, and opacifiers/pigments mainly composed of antimony-based compounds typical of Roman tradition, indicate the extensive availability of older glass for re-use and/or a preference for recycling, rather than the preparation of new batches. Peculiar is also the finding, in one Vicenza Aquamarine sample, of an opacifier composed of sodium antimonate, never found in ancient glass tesserae until now. Comparisons between the Vicenza and Padova mosaics indicate technological connections between the sites, as testified by the many chromatic groups with high degree of comparability. However, when differences are observed into chromatic groups compared, the Vicenza tesserae generally show older technological features than those of Padova. The hypothesis advanced here, related to the different geographical location of the two sites, explains such experimental evidence in terms of the different speeds at which innovative technologies spread in north-eastern Italy.