The aluminum (Al) tolerance of hexaploid wheat and rye has been extensively studied. However, the Al tolerance of tetraploid wheat (Triticum turgidum L.), the AABB genome donor of hexaploid wheat, has not been well characterized. To understand the Al tolerance of tetraploid wheat and identify potentially new sources of tolerance to acidic soils, 254 Triticum turgidum wheat lines, including six subspecies of T. turdigum ssp. dicoccoides, ssp. dicoccon, ssp. carthlicum,ssp. turgidum, ssp. durum, and ssp. polinicum, were screened for acid and Al tolerance by measuring the root regenerate lengths and the tolerance indices in acid nutrient culture solution containing Al. Significant differences were observed either among or within tetraploid wheat subspecies on the RRL (root regenerate lengths). The orders for average RRL among subspecies in pH 7.00, pH 4.50 and pH 4.50 + 50 μmol L-1 Al3+ were carthlicum ≥ turgidum ≥ durum ≥ dicoccoides ≥polinicum≥dicoccon, carthlicum ≥ turgidum ≥ durum ≥ polinicum ≥ dicoccon ≥ dicoccoides and durum ≥ dicoccoides ≥carthlicum ≥ turgidum ≥ polinicum ≥ dicoccon, respectively. Significant difference was also observed on the root acid tolerance index (RTI1) and root Al tolerance index (RTI2) either among or within tetraploid wheat subspecies. Compared to RTI2, RTI1 varied in a wide range but with large value. The orders of the average RTI1 and RTI2 among subspecies were polinicum ≥ dicoccon ≥ carthlicum ≥ dicoccoides ≥ turgidum ≥ durum, and dicoccoides ≥ durum ≥ turgidum ≥ polinicum ≥ carthlicum ≥ dicoccon, respectively. Significant difference on acid and Al tolerance was existed either among or within subspecies. Some potentially tolerance germplasms different from those derived from hexaploid wheat, including 42 acid tolerance and 41 Al tolerance lines, were identified in 254 tetraploid wheat lines. They provide new sources for hexaploid wheat acid and Al tolerance improvement.