Clarifying the pedigree on Eucalyptus pellita populations is of great significance for studying rules of interspecific hybridization of eucalypt and the development of excellent new eucalypt genotypes. The purpose of the present study was to assess the accuracy and reliability of near infrared spectroscopy (NIRs) used in the analysis of the pedigree of E. pellita populations by comparing the relationship between genetic variations and NIRs differences that. The genetic materials involved natural provenances from the E. Pellita population, fresh leaves of 8~12 families were collected from each provenance. The DNA information of materials was obtained through whole-genome resequencing. Firstly, the genetic distances among provenances were evaluated with the DNA nucleotide sequence differences between samples. Meanwhile, four to six healthy leaves of each sample were placed in a drying ovenuntil completely dry. The dried leaves were milled and then put into a transparent self-sealing plastic bag. A portable NIR device, phazir RX (1 624), was used to take the NIRs information of samples. The NIRs spectral distance between validating provenance and calibrating provenance was estimated with the soft independent modeling of class analogy (SIMCA). Hierarchical clustering was performed for all provenances with NIRs Euclidean distance. PCA scores plots of provenances NIRs demonstrated the pedigree and the genetic variations of provenances. The results showed that the total mean of the genetic distance of provenances from New Guinea Island and Queensland were 0.186 and 0.157 respectively, the total mean of genetic distance between New Guinea Island and Queensland was 0.295, which was higher than that within each separate district significantly. There was a positive correlation between NIRs spectral distance and genetic distance between provenances in two separate districts, but a negative correlation was also found between some provenances of E. pellita. The correlation between genetic distance and NIRs spectral distance was also proved by the NIRs Hierarchical clustering of all provenances. However, the clustering did not completely correspond with their geographical distance of provenances, suggesting that gene flow of some forms greatly affects the genetic relationship among separate districts of E. pellita populations. The PCA score plots demonstrated that PCs plots of some provenances with large genetic distance or NIRs spectral distance would overlap seriously, and PCs plots of some provenances with close genetic distance or NIRs spectral distance would be clustered, which verified the sensitivity of NIRs in the distinguishing of heterogeneous samples, also showed the genetic variation among families inprovenance of E. pellita. All the current study results proposed that NIRs could genuinely reflect the genetic differences among provenances of E. pellita, and could be used to analyze the genetic relationship and genetic variation within eucalypt populations, and could be used to assist the improvement of eucalypts breeding populations in a generation.