Land-use change for bioenergy production can release greenhouse gases (GHG) through disturbance of soil carbon (C) pools, but use of native species with extensive root systems as bioenergy crops may help mitigate GHG emissions by enhancing soil C sequestration. Here, we investigated how (1) fertilization, (2) plant species and cultivars, and (3) inter- and intra-specific diversity affect soil C and N accumulation five growing seasons after conversion of an old-field dominated by C<
sub>
3<
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grasses to a grassland dominated by C<
sub>
4<
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perennial grasses managed for biofuel production. We manipulated diversity at both the species- and cultivar level, and applied nitrogen (N) at two levels (0 and 67 kg ha<
sup>
?1<
/sup>
). Establishment of C<
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4<
/sub>
grass treatments on soils that supported C<
sub>
3<
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pasture grasses for 36 years enabled us to use the natural abundance C isotope ratio technique to estimate the contribution of new C<
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4<
/sub>
plant-derived C to soil organic matter pools. Our study yielded three main results: 1) annual fertilization did not significantly affect soil C and N concentrations after five growing seasons
2) increasing inter- and intra-specific diversity did not significantly increase soil C and N concentrations
3) cultivar- and species identity influenced C<
sub>
4<
/sub>
-derived C and total soil C concentrations: big bluestem dominated stands exhibited greater soil C accrual relative to stands dominated by switchgrass and mixed-species treatments. Here, future research is needed to further assess how big bluestem can aid in the sustainable provisioning of second generation biofuel feedstocks.