MassTag polymerase chain reaction (PCR) is a platform that enables microbe detection using primers labeled through a photocleavable link with tags that vary in molecular weight. After multiplex PCR, tags are released by ultraviolet irradiation and analyzed by mass spectroscopy. The identification of a microbe in a sample is determined by its cognate tags. Here we describe establishment and implementation of a MassTag PCR panel for surveillance of microbes implicated in tick-vectored infectious diseases.