Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) continues to be a major problem to the pork industry worldwide. The limitations offered by current PRRSV vaccines require the development of a new generation of vaccines. One of the key steps in future vaccine development is to include markers for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. In previous study, we have constructed a marker virus, which contains a green fluorescent protein (GFP) insertion and an immunogenic epitope deletion in nsp2 region of the virus. In this study, we performed in vivo characterization of this marker viruse. To compliment the marker identification, we developed GFP and nsp2 epitope-based ELISAs. Pigs immunized with the recombinant virus lacked antibodies directed against the corresponding deleted epitope, while generating a high level of antibody response to GFP by 14 days post-infection. Our results demonstrated that this recombinant marker virus, in conjunction with the diagnostic tests, enable serological differentiation between marker virus-infected animals from those infected with the wild-type virus. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to assist with the control of PRRSV.