Management factors for enhancing C sequestration in agricultural ecosystems involve increasing crop residue inputs and decreasing soil organic matter decomposition. The use of 13C natural abundance techniques, coupled with physical and/or chemical fractionation, has provided additional insight into soil organic matter turnover for cases where appropriate changes in vegetation from C3 to C4, or vice versa, have occurred. Our study evaluated effects of cropping sequence and tillage on 13C and 15N in physically- and chemically-separated soil organic matter pools. The 13C in all fractions was primarily influenced by crop species rather than tillage. In continuous wheat, the natural abundance of 13C decreased in the order of silt and clay fraction>resistant organic matter > microaggregates > particulate organic matter. Continuous sorghum showed a reverse trend as it is a C4 crop. Tillage effects on 13C were primarily observed in labile pools (protected and unprotected particulate organic matter). The 15N values were lowest in resistant and particulate organic matter pools and highest in the microaggregate fractions. In general, 15N contents were greater under no tillage than conventional tillage regardless of cropping sequence. Our study indicated that natural abundance of 13C and 15N was a useful tool for examining effects of cropping sequence and tillage on soil organic C and N distribution in physical and chemical organic matter pools.