Siraitia grosvenorii was an important Chinese traditional medicine. The spectra of leaf of diploid, triploid and tetraploid Siraitia grosvenorii were determined by Fourier transform infrared spectroscopy (FTIR) with OMNI sampler directly, fast and accurately. And based on the indices of wave number-absorbance from different bands and through comparison of differences of these infrared spectra the ploidy difference and genetic relationship of Siraitia grosvenorii were studied by the methods of principal component analysis (PCA) and cluster analysis. The results showed that for the ploidy, the position relationship of PCA three-dimensional-plot and the distance coefficient of clustering analysis plot between diploid and tetraploid were most remarkbly different, and the triploid was basically between diploid and tetraploid, so the ploidy was more different, the position relationship of PCA and the distance coefficient of clustering analysis were further, and the law was: 2×<3×<4×. At the same time, the genetic relationship was further with each other while the position relationship of PCA and distance coefficient of clustering analysis was further too. And the genetic relationship of triploid was affected by the different male parent while their female parent was the same one. Therefore, using FTIR based on PCA and cluster analysis we could reveal the difference of ploidy and the genetic relationship between Siraitia grosvenorii to a certain extent. FTIR could be used for excellently polyploidy species breeding of Siraitia grosvenorii.