Background/Question/Methods Non-bloat legume forages such as cicer milkvetch (Astragaluscicer) and sainfoin (Onobrychisviciifolia) can be an alternative for bloat legume - alfalfa (Medicagosativa) in pasture systems to improve feed use efficiency and reduce enteric methane emissions. However, there is no information on changes in root-derived carbon allocation and microbial utilization for these alternative legume species. A13C/15N dual pulse-labeling greenhouse experiment included three binary mixtures [either alfalfa, cicermilkvetch or sainfoin] grown together with bromegrass(Bromusmadritensis)]. The experiment consisted of 8 isotopically-labelled treatments in 4 replications of 3 sets: legume labelling (3), bromegrass labelling (4) and bare soil (1). The non-labelled control treatments were 3 legume-bromegrass mixtures, bromegrass alone, and bare soil. Plants were were separately cross-labeled using 13CO2 99atom%13C and 15N Urea 98atom%15N 0.2 % v/v, started 1 month after transplanting. 13C labelling was implemented twice a week for the first week and once a week for the following3 weeks, resulting in 5 labelling sessions. 15N labelling was kept continuously for 4 weeks, in total of 4ml 15N Urea solution. Soils were sampled at 1 and 7 days after the last labeling event and were be analyzed for root-derived 13C, 15N, and 13C stable isotopic phospholipid fatty acid analysis (13C-PLFA) to link microbial communities with root-derived carbon and nitrogen estimation. Results/Conclusions Results showed that while root derived N at day 1 (0.56 – 2.11 mg N) were lower than at day 7 after last labeling (ranged from 0.65 – 3.48 mg N) in all binary mixtures , the trend for bromegrass growing alone was reversed (1.85 vs 1.39 mg N). Root derived C experienced the opposite trend with root derived N. CdfR at day 1 (19.3 – 42.7 mg C) were higher than at day 7 after last labeling (ranged from 12.4 – 28.5 mg N) in all binary mixtures. In bromegrass alone, it was increased from 40.8 mg C (day 1) to 45.8 mg C (day 7). 13C-PLFA results will determine differences in active microbial groups (i.e., gram-negative/gram-positive bacteria, arbuscular mycorrhizal fungi, saprophytic fungi) that are involved in the short-term processing of root-derived resources from different legume-bromegrass plant combinations.