Sub-cellular proteomics provide insight into the molecular mechanisms of plant cell modulation of protein accumulation in intracellular compartments regarding various perturbations, and thus provides rectified knowledge about signal transduction in organelles. Mitochondria are important organelles for cellular respiration within the eukaryotic cell and serve many important functions including vitamin synthesis, amino acid metabolism and photorespiration for the cell as well. To define the mitochondrial proteome of the roots of wheat seedling, a systematical and targeting analysis were carried out on the mitochondrial proteome from 15 days-old wheat seedling roots material. Mitochondria were isolated by Percoll gradient centrifugation; and extracted proteins were separated and analyzed using Tricine SDS-PAGE along with LTQ-FTICR mass spectrometry. From the isolated mitochondrial proteins, a total of 140 proteins were identified. The identified proteins were functionally classified into 12 classes using ProtFun 2.2 server based on cellular roles, Proteins were shown to be involved in including amino acid biosynthesis (17.1%), biosynthesis of cofactors (6.4%), cell envelope (11.4%), central intermediary metabolism (10%), energy metabolism (20%), fatty acid metabolism (0.7%), purines and pyrimidines (5.7%), regulatory functions (0.7%), replication and transcription (1.4%), translation (22.1%), transport and binding (1.4%), and unknown (2.8%). These results indicated that many of the protein components present and functions of identified proteins are common to other profiles of mitochondrial proteomes performed to date. The data presented here will begin to reveal a better understanding the characteristics of proteins and metabolic activity in mitochondria in wheat roots.