Al toxicity in acidic soils is a major constrain for crop production worldwide. Malate release from root tip has been considered as a major mechanism of Al resistance in wheat. Our previous study has identified new Al-resistant germplasm carrying the sensitive marker allele of ALMT1 locus, and suggested possible new genetic mechanism may be involved in Al resistance of those cultivars. In this study, the expression level of ALMT1 was quantified in selected wheat cultivars using real-time PCR (RT-PCR) to validate the marker data. The results from RT-PCR indicated that the differences in the levels of ALMT1 expression were not significant between Al-stressed resistant plants and unstressed resistant controls, but significant between Al-sensitive and most of Al-resistant cultivars, indicating ALMT1 was constitutively expressed in most Al-resistant wheat cultivars, but not in any susceptible cultivars. However, a few Al-resistant cultivars such as Chinese landrace FSW carry the sensitive marker allele of ALMT1 and showed a low level of ALMT1 expression as Al-sensitive cultivar Century, suggesting that expression of ALMT1may not be responsible for Al resistance in FSW. Malic acid was quantified from the culture solution and the root tips of cultivars Atlas 66, Century and FSW after two days of Al treatment.  Significant larger amount of malate was detected from culture solution and root tips of Atlas 66, than that from Century. A high level of malate was detected only in root tips of FSW only when the seedling was treated with a high Al concentration.  Malate concentration in cultural solution or root tips of FSW was similar to Century when seedling was treated with a low Al concentration. Therefore, the expression of ALMT  may not essential for Al resistance in some wheat genotypes and  other mechanisms for Al resistance may exist in wheat.