Fluorescent dyes are an indispensable part of the scientific enterprise. Xanthene-based fluorophores, like fluorescein and rhodamine, have been in continual use across numerous fields since their invention in the late 19th century. Modern methods to synthesize and expand the scope of xanthene dye chemistry have enabled new colors, enhanced stability, and improved brightness. Modifications to the 3-position of xanthene dyes have been, until recently, less well-explored. Here, we discuss how small changes to the identity of the substituent at the 3-position of fluoresceins and rhodamines can profoundly alter the properties of xanthene dyes, with the potential to unlock new applications at the interface of chemistry and biology.