Chlorophyll biosynthesis is catalyzed by a series of enzymes. Among them, the chlorophyll synthase (CHLG) catalyzes the synthesis of chlorophyll a and b, the last step of the pathway. In this study, Medicago truncatula (ecotype R108) and Medicago sativa with fall dormancy of 4 or 8 were used. The evolutionary relationship of plant CHLGs and their gene structure were analyzed using bioinformatics; the expression of MtCHLG under light/darkness, abscisic acid (ABA) or polyethylene glycol (PEG) treatment was studied by realtime quantitative PCR; the transcript level of MtCHLG was tested in mtcao mutant; and the correlation coefficient between the expression level of MsCHLG and plant height or yield in Medicago sativa was analyzed. The results showed that: (1) CHLGs shared common conserved domains and a relatively uniform gene composition. It seems that several terrestrial plants possess one more CHLG with much longer introns. (2) MtCHLG open reading frame (ORF) of 1 137 bp was cloned encoding 378 amino acids and the protein was highly identical (99.2%) to MsCHLG. (3) qRTPCR demonstrated that MtCHLG was predominantly expressed in young leaves of Medicago truncatula with a lightinducible or darkrepressed pattern showing a significant increase in the daytime and a continuous decline at night. Moreover, under 100 μmol·L^-1 ABA treatment of 2 h, MtCHLG in leaves decreased to about 26% of the control (P<0.05). 5% PEG treatment within 24 h repressed the expression of MtCHLG, which decreased to about 42% of the control (P<0.05) at the end of the treatment. (4) When transiently expressed in tobacco epidermal cells, the recombinant protein MtCHLGGFP was detected mainly in chloroplasts. (5) qRTPCR showed that in mtcao mutant with no obvious expression of MtCAO, the expression level of MtCHLG was about 44% of the wild type, indicating that MtCAO lossoffunction resultes in a decrease of MtCHLG, which suggests that MtCHLG is downstream of MtCAO in the chlorophyll biosynthesis pathway. (6) The plant height of the alfalfa accessions with FD of 8 was about 2.5-3.5 times of the ones with FD of 4. The average yield of the former was about 2.1 times of the latter, and the expression level of MsCHLG showed a similar pattern (1.2-1.5 times). According to the correlation coefficient, the expression level of MsCHLG was significantly correlated with both plant height and yield. The findings suggest that MsCHLG transcriptional level might be useful for preliminary prediction of alfalfa yield at early stage, and CHLG may serve as a candidate gene to improve yield of legume forages by breeding.