The diffuse reflection and diffuse transmission spectra of 491 cocoon samples were collected and compared for male and female discrimination. Maya2000pro NIR spectrometer was used in this experiment. Subsequently, diffuse transmission spectra within 450~1050 nm was approached by the mean center and the first order derivative pretreatment. Finally, partial least squares discriminant analysis (PLSDA) was used to make a predictive analysis for the three species of cocoon samples. As a result, the diffuse transmission spectra were better to reflect the characteristics of the components inside the chrysalis, compared with the diffuse reflective spectra. The diffuse transmission spectra could distinguish male and female well respectively for three species (sensitivity of 89.796%, specificity of 92.424% and accuracy of 90.854% for variety Shidong A〖DK〗·Hua 3xC〖DK〗·7532; and sensitivity of 96.250%, specificity of 94.253% and accuracy of 95.210% for variety Huazhong 2〖DK〗·Hua 3xC〖DK〗·7532; sensitivity of 97.260%, specificity of 91.954% and accuracy of 94.375%for variety 9〖DK〗·Fu x7〖DK〗·Xiang ). The identification results of all samples mixed together were also good with sensitivity of 94.024%, specificity of 92.917% and accuracy of 93.483%.