Abstract The purpose of this study is to determine whether urban greenhouse gas (GHG) fluxes can be quantified from tower-based mole fraction measurements using Monin–Obukhov similarity theory (MOST). Tower-based GHG mole fraction networks are used in many cities to quantify whole-city GHG emissions. Local-scale, micrometeorological flux estimates would complement whole-city estimates from atmospheric inversions. CO2 mole fraction and eddy-covariance flux data at an urban site in Indianapolis, Indiana, from October 2020 through January 2022 are analyzed. Using MOST flux–variance and flux–gradient relationships, CO2 fluxes were calculated using these mole fraction data and compared to the eddy-covariance fluxes. MOST-based fluxes were calculated using varying measurement heights and methods of estimating stability. The MOST flux–variance relationship method showed good temporal correlation with eddy-covariance fluxes at this site but overestimated flux magnitudes. Fluxes calculated using flux–gradient relationships showed lower temporal correlation with eddy-covariance fluxes but closer magnitudes to eddy-covariance fluxes. Measurement heights closer to ground level produce more precise flux estimates for both MOST-based methods. For flux–gradient methods, flux estimates are more accurate and precise when low-altitude measurements are combined with a large vertical separation between measurement heights. When stability estimates based on eddy-covariance flux measurements are replaced with stability estimates based on the weather station or net radiation data, the MOST-based fluxes still capture the temporal patterns measured via eddy covariance. Based on these results, MOST can be used to estimate the temporal patterns in local GHG fluxes at mole fraction tower sites, complementing the small number of eddy-covariance flux measurements available in urban settings.