A defining characteristic of the planet Venus is its thick, CO2-dominated atmosphere. Despite over fifty years of robotic exploration, including thirteen successful atmosphere probes and landers, our knowledge of N2, the second-most-abundant compound in the atmosphere, is highly uncertain (von Zahn et al., 1983). We report the first measurement of the nitrogen content of Venus atmosphere at altitudes between 60 and 100 km. Our result, 5.0 +/- 0.4 v% N2, is significantly higher than the value of 3.5 v% N2 reported for the lower atmosphere (<50 km altitude). We conclude that Venus atmosphere contains two chemically-distinct regions, contrasting sharply with the expectation that it should be uniform across these altitude due to turbulent mixing (e.g. Oyama et al., 1980). That the lower-mass component is more concentrated at high altitudes suggests that the chemical profile of the atmosphere above 50-km altitude reflects mass segregation of CO2 and N2. A similar boundary between well-mixed and mass-segregated materials exists for Earth, however it is located at a substantially higher altitude of ~100 km. That Venus upper and lower atmosphere are not in chemical equilibrium complicates efforts to use remote sensing measurements to infer the properties of the lower atmosphere and surface, a lesson that also applied to the growing field of exoplanet astronomy. The observation of periodic increases in SO2 concentrations in Venus upper atmosphere, which has been cited as evidence for active volcanic eruptions at the surface (Esposito et al., 1984), may instead be attributable to atmosphere processes that periodically inject SO2 from the lower atmosphere into the upper atmosphere.