Objective:The matria-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS) and sequencing methods were performed to assess the methodology (biochemistry methods) for identifying the Pandoraea sputorum and provide the more preferred approach to identify Pandoraea species. Methods:This paper is a study on performance evaluation of identification method. Ten lines of Pandoraea sputorum were isolated from blood cultures of inpatients were collected at Zhongnan Hospital of Wuhan University from July to October 2018 and were confirmed by the cultural characteristics, colonial morphology and Gram′s stain. Further identification was carried out by using the manual biochemical method (API 20NE), automatic biochemistry systems(BioMerieuxVITEK 2 Compact, BD Phoenix-100andThemo ARIS 2X), MALDI-TOF MS (BioMerieuxVITEK MS and Bruker MALDI Biotyper) and the sequencing methods of the 16S rRNA to identify the Pandoraea sputorum. Results:Pandoraea sputorum was non-fermented gram-negative bacteria that are non-motile, oxidase positive, and catalase positive. Ten lines of Pandoraea sputorum were identified as Achromobacter denitrificans, Alcaligenes faecalis or Cupriavidus pauculus and the accuracy rate of genus and species identification was 0 by API 20NE. Among all the samples, six lines were identified as the Pandoraea spp. with the accuracy rate of genus identification was 6/10 by VITEK 2 Compact; whereas the other four lines were identified as the Burkholderia cepacia, Sphingomonas paucimobilis, Ralstonia pickettii, or "Low Discrim" . All of these were identified as "No Identification" by Phoenix-100, which the accuracy rate of genus and species identification was 0. Seven isolates were identified by ARIS 2X as Stenotrophomonas maltophilia, Acinetobacter lwoffii, Sphingomonas paucimobilis, Pseudomonas luteola, Acinetobacter baumannii/Acinetobacter haemolyticus; whereas the other three lines were identified as rare species, thus, the accuracy rate of genus and species identification was 0. Both VITEK MS and MALDI Biotyper indicated all the isolates were Pandoraea sputorum with the accuracy rate of genus and species identification was 10/10. 16S rRNA sequencing for the 10 isolates showed they have 100% of similarity to Pandoraea sputorum by blasting with Genebank. Conclusions:Methods based on biochemical reactions often failed to accurately identify the Pandoraea sputorum to species level. MALDL-TOF MS and 16S rRNA sequencing technology identify Pandoraea sputorum efficiently and precisely enough.