Most currently available HIV-1 reporter gene constructs are large and disrupt the nef reading frame. This report describes a novel reporter gene based on the small murine heat stable antigen (HSA) protein, which is expressed on the surface of infected cells. This HSA reporter can be inserted in the vpr reading frame, leaving nef intact. Nine amino acids from the extracellular domain of HSA are replaced with an influenza hemagglutinin (HA) antibody epitope (HSA-HA). Like the parental reporter protein, this novel reporter is expressed on the surface of infected cells. Antibodies for HSA and HA specifically detect reporter viruses with each construct, indicating disruption of the original HSA antibody epitope. Finally, a strategy is developed to detect each reporter virus by real-time PCR quantitation. The growth of viruses tagged with each reporter allows precise assessment of the relative growth of viruses differing in mutations of interest. Moreover, the availability of these reporters in either of two half-genome plasmids allows convenient production of reporter and non-reporter HIV-1 by co-transfection of appropriately paired plasmids. These paired reporter viruses offer a potentially useful standardized method for measurement of HIV-1 fitness in competition assays.