Lespedeza speices can grow very well in infertile acidic soils as native pioneer plants. The reasons why such plants are well adapted to these soils might be due to a combination of mechanisms. Al toxicity is the primary factor limiting plant growth in acidic soils. Our previous results indicated that secretion of malate and citrate from roots was related to the high Al-resistance of Lespedeza. The objective of this study was to investigate the formation of callose under Al stress and its regulating factors in two Lespedeza species with different Al resistance, and attempted to explore the relative role of callose formation and organic acid exudation in Al toxicity or resistance of Lespedeza. The results showed that increased Al uptake was associated with increased callose formation in both Lespedeza species. The differences in Al tolerance between the two species were mainly expressed at the root apical 0-0.5 cm. Moreover, the callose formation was similar to root Al uptake for Al-sensitive species, but not for Al-tolerant species, of which callose formation peaked at 6 h of Al treatment and then decreased. Callose formation of the tolerant species was not obviously reduced following removal of Al from the culture. Furthermore, no obvious differences in callose formation were observed under treatment with PG (phenylglyoxal, anion-channel inhibitor) plus Al as compared to Al treatment alone in both species. NIF (niflumic acid, anion-channel inhibitor) plus Al treatment inhibited callose formation in the Al-sensitive species, but not in the Al-tolerant species. Callose formation was inhibited by A-9-C (9-anthracene carboxylic acid, anion-channel inhibitor) plus Al treatment in both species. Compared with Al treatment alone, DDG plus Al treatment caused a significant reduction in callose formation in the Al-sensitive species but not in the Al-tolerant species. No differences in callose formation were observed for both species following mannitol treatment. Callose formation in response to La treatment was significantly higher in the Al-tolerant species than in the Al-sensitive species and was still higher following treatment with both Al and La. However, no differences in callose formation in Al-sensitive species were detected among treatments. In summary, callose synthesis may be regulated in conjunction with organic acid exudation. Callose formation may be a sensitive indicator of Al stress and may also function in Al tolerance before organic acids are secreted from roots. Finally, callose formation may be dependent on many factors, including Al concentration, treatment time, organic acid exudation, and osmotic pressure. Further studies are required to examine these additional factors to determine the complex mechanisms mediating callose formation.