Soil organic carbon (SOC) content is a key metric of soil quality and limited work has been done to examine the effect of long-term conservation agriculture management practices (CAMP) on SOC pools within western Canadian soils. We assessed the nature and permanence of sequestered SOC within 90 diverse Saskatchewan surface (0–10 cm) agricultural soils before and after 21 years of CAMP. Comparisons were made of total SOC, labile and dynamic SOC fractions (light fraction, water-extractable, and microbial biomass), respirable CO2-C during a six-week incubation, along with spectroscopic characterization using 13C/12C stable isotope ratio and ATR-FTIR. Among soil climatic zones, the SOC content increased in the semi-arid Brown and Dark Brown soils, ranging from 2.4–3.7 Mg C ha-1 (111.4–187.7 kg C ha-1 year-1), but did not change in the subhumid Black, Dark Gray, and Gray soils. Overall, soils having the smallest initial SOC level were most responsive to CAMP and accumulated more SOC. According to the δ13C data, CAMP appeared to reduce annual crop moisture stress, especially within the Brown soil zone. Decreased light fraction and water-extractable SOC contents in Black, Dark Gray, and Gray soils could reflect more intense decomposition and greater surface stratification of crop residues. Brown soils experienced the largest increase in microbial biomass-C content. The CO2-C emissions from the Brown, Dark Brown, and Gray soils under CAMP suggest greater SOC stability in 2018 compared with 1996. The ATR-FTIR data pointed to enhanced SOC persistence, via more stabilized SOC forms and mineral-associated organic C fractions.