The genetic backgrounds of many Citrus varieties are quite complex. Classifications and phylogenetic relationships of Citrus species have become the focus of researchs. Some conserved genes of chloroplast genome's research have been proven effective in determining the biosources of hybrids and phylogenetic analysis. Thus, we studied variations among the chloroplast trnL gene sequences of 10 Citrus species, including C. nobilis Lour. 'Gonggan'. The amplification results of different trnL target genes and identification of the double-enzyme cut after cloning show that lengths of all trnL sequences were within 895 to 935 bp and a total of 24 variation sites were detected among the 10 material samples. Clustering analysis revealed differences in trnL genes caused by systematic evolution and allowed the determination of variations among Citrus varieties. Variation sites of trnL sequences can be used in the phylogenetic classification and species identification of Citrus, and the results agreed with random amplified polymorphic DNA marker results. C. nobilis Lour. 'Gonggan' is closely associated with the other two varieties in Zhaoqing area, and C. nobilis Lour. 'Gonggan' and C. haniana Hort. ex Tseng 'Sihuihanggan' can be classified into the same category. C. nobilis Lour. 'Gonggan' as a natural hybrid is probably a hybrid with C. haniana Hort. ex Tseng 'Sihuihanggan' as its female parent.