The relationship between soil heterotrophic respiration (Rh) and soil moisture conditions has been often studied using disturbed soil samples and simple gravimetric and volumetric soil moisture indicators. The objective of this study was to investigate the relationship between Rh and soil moisture in terms of water-filled porosity (WFP), matric potential (), and relative soil gas diffusivity () using undisturbed soil cores obtained under different land covers. Soil CO2 efflux, WFP, and were simultaneously measured in undisturbed soil samples (250 cm3) without collected in the 0-5 cm soil layer without any vegetation or living roots under laboratory conditions by combining a CO2 gas analyzer, a precision scale, and precision mini-tensiometers. For each sample we also measured soil chemical properties, soil physical properties, and soil microbial composition using phospholipid fatty acid analysis. Grasslands had the largest total microbial biomass (6,275 ng g-1), followed by soils from riparian (5,327 ng g-1), and cropland (2,745 ng g-1) sites. Bacteria were the dominant group representing 46% (SD = 5%) of the total biomass across all sites and land covers. Maximum Rh was 1.88 (SD = 0.40) μmol CO2 m-2 s-1 in grassland, 1.64 (SD = 0.82) μmol CO2 m-2 s-1 in riparian, and 0.94 (SD = 0.56) μmol CO2 m-2 s-1 in cropland soils. Using WFP as the predictor variable resulted in the most consistent moisture response function. Considering all land cover and soil types, our observations revealed that peak Rh occurred at mean WFP = 0.81 = -6 kPa, and = 0.003. The simpler WFP soil moisture indicator was a better predictor of Rh than or from air-dry to near saturation across soils with contrasting soil texture and soil structure.