Chondroitin sulfate (CS) is a glycosaminoglycan playing several biological functions, which seem to be encoded through its sulfation pattern. This "sulfation code" is still to be deciphered. One of the barriers to this goal is the difficulty in achieving structurally well-defined CS polysaccharides since extraction from natural sources often leads to complex heterogeneous structures. Instead, an approach relying on chemical modification of a microbially sourced unsulfated chondroitin can allow access to semisynthetic CS polysaccharides with a well-defined sulfation pattern. We report herein some new, suitably developed chemical strategies affording CSs with unprecedented sulfation patterns, carrying a single sulfate group regioselectively placed at either C-2 or C-3 position of the glucuronic acid residues or at both sites. In this way, all the possible variants of CS sulfation patterns can be now accessed. This will allow more detailed and complete structure-activity relationship investigations of CS biological functions and applications.