Adapting agriculture to long-term accrual of organic carbon (С) is beneficial both for ensuring food security and for mitigating climate change. This study quantified the responses of total soil C content and its constituent pools to implementing no-tillage (NT) versus conventional tillage (CT) on farms with contrasting water regimes. The farms were chosen at two sites in the Russian steppe zone: Rostov with non-waterlogged Calcic Chernozem (CCH; sunflower-wheat rotation) and Krasnodar with periodic waterlogged Stagnic Chernozem (SCH; maize-wheat rotation). At each site, we surveyed the 0–10 cm and 10–30 cm soil layers in one continuous CT field and two short-term NT fields (8–14 years). The average C content in CCH was higher than in SCH (22.5 vs 17.7 g kg–1). For both sites, NT showed the potential for an increase in C content (by 12–16%) relative to CT only in the 0–10 cm topsoil. Microbial-available C pool (mineralized for 180 days of soil incubation) was most sensitive to tillage systems, unlike unchanged particle-size pools. Specifically, it increased from CT to NT for CCH (by 7–16%), but it showed a decreased trend for SCH (by 11–29%), possibly due to the worsening of soil aeration in the periodically flooded regime. Gradient boosting machine models accurately predicted the spatial distribution of topsoil C content (R2 = 0.99) and its microbial-available pool (R2 = 0.78%) across the farmland area. The mutual drivers of both parameters were topography (elevation) and vegetation distribution (near-infrared surface reflectance). These outcomes are useful for developing site-specific management strategies to effectively restore C stocks in Chernozem soils.