Soil properties, plant characteristics, agronomic practices and environmental factors often influence biological nitrogen fixation of legumes. Tillage method used in a specific agro-ecological zone is among these factors thus, the aim of this study was to determine the effect of tillage and no tillage on biological nitrogen fixation and grain yields of three soybean varieties. The study was conducted at four agro-ecological zones of Western Kenya where treatments were replicated thrice using randomized complete block design in split plot arrangement. No tillage and conventional tillage were the main plots whereas, soybean varieties: Nyala, SB19 and SB20 were the sub-plots. Amount of N fixed was determined using 15N abundance method and quantity of nitrogen fixed under no tillage practice exceeded conventional tillage at all sites however, soybean varieties fixed same amount of N. Interaction effect of tillage method and variety on amount of N fixed was varying at every agro-ecological zone though, interaction between no tillage and Nyala fixed more nitrogen at LM 3 than at other agro-ecological zones. Soybean grain yield was similar between tillage methods and also among varieties in a combination of all sites. Practicing no tillage encourages biological nitrogen fixation and its longtime operation leads to yield increase as it improves most soil properties.