The molecular weight (Mw) of hyaluronic acid (HA) determines its suitability for medical and cosmetic applications. Here, we characterize in vitro and in vivo HA synthesis of streptococcal HA synthases (HASs) with a special focus on HA Mw. To date, four streptococcal HA producers are described (Streptococcus equi subsp. equi, S. equi subsp. zooepidemicus, S. pyogenes, and S. uberis). We identified two more potential HA producers in this study: S. iniae and S. parauberis. Indeed, the HA Mw produced by the different streptococcal HASs differs in vitro. To exploit these different HA Mw synthesis capacities, Lactococcus lactis strains expressing the streptococcal HASs were constructed. HA of different Mw was also produced in vivo by these engineered strains, strongly suggesting that the protein sequences of the HASs influence HA Mw. Since the HA Mw in vivo is also influenced by metabolic factors like specific growth rate and HA precursor availability, these were also determined. In summary, the maximal Mw of HA synthesized is specific for the individual synthase, while any decrease from the maximal HA Mw is influenced by physiological and metabolic factors. The results open new avenues for Mw-tailored HA synthesis.