The silk trait secreted by Bombyx mori posterior silk glandule (PSG) consists of three polypeptides: fibroin heavy (H) and light (L) chains, and the accessory protein P25. The fibroin L-chain (fib-L) gene is 13472 bp long, containing seven exons with great introns inserted. The first intron consists of about 60% of the gene length while the other introns together only represent approximately 31% of it, therefore, no codante sequences represent 91% of the gene, which is an important gene for a suitable silk trait in commercial cocoon. In this work, we analyzed the nucleotide variability in the fib-L third intron amongst different B. mori strains. The experimental procedure was based in the analysis of three individuals of each strain: the Japanese HA-B and the Chinese C121-A and C122-B. Genomic DNA was extracted and amplified with primers based in the third and fourth exons. The 903 bp product was characterized through Conformation Sensitive Gel Electrophoresis (CSGE) method and sequenced using DyEnamic ET Dye Terminator in MegaBACE 1000 automatic sequencer. The analysis of third intron sequences of fib-L gene was performed using Phred, Phrap, Consed, BioEdit, ClustalW 1.8 and PAUP software. The phylogenetic study was performed using the Maximum Likelihood (ML) method whit 1000 replicas bootstrap and visualized in the Treeview software, using two sequences available in the GenBank, M76430 (Japanese) and AF541967 (hybrid), for comparative analysis. In the multiple alignments of all sequences it was possible to observe 15 conserved regions from 12 to 61 bp long that represent 51.60% of the intron sequence. The results indicate two clusters in the phylogenetic tree topology. The first cluster is composed by the Japanese strains: HA-B (individuals 1 and 3), M76430 and the Chinese C122-B (individual 1). The second cluster is formed by the Chinese strains: C121-A (all three individuals), C122-B (individuals 2 and 3) and the hybrid AF541967. The presence of the Chinese C122-B (individual 1) strain in the first cluster was an unexpected result, and in the second group the other two individuals of this strain showed a significant genetic distance with bootstrap value of 99%. Apart from the Chinese C122-B results, the screening of the Bombyx mori third intron fib-L gene variability is a powerful tool for silkworm strains characterization.