ObjectiveTo investigate genotypes of extendedspectrum βlactamases（ESBLs） produced by Escherichia coli （E. coli） and Klebsiella pneumoniae (K. pneumoniae) isolated from a hospital.MethodsClinical isolates of ESBLsproducing E. coli (n=40) and K. pneumoniae (n=20) were collected according to ESBLs confirmatory test. Plasmid DNA was extracted by alkaline lysis method. PCR analysis was used to amplify TEM, SHV, and CTXM genes, sequence analysis was performed to identify βlactamase genotype.ResultsSixty confirmatory test positive E.coli and K.pneumoniae were positive for PCR amplification, including TEM,SHV, CTXM3 and CTXM9. The positive rate of above four genotypes among E.coli was 37.50%（15 isolates）,2.50%（1 isolate）,62.50%（25 isolates） and 50.00%（20 isolates）respectively; the positive rate of above four genotypes among K.pneumoniae was 40.00%（8 isolates）,90.00%（18 isolates）,65.00%（13 isolates） and 40.00%（8 isolates）respectively. 100.00% of E.coli and 80.00% of K.pneumoniae expressed blaCTXM, 12.50% (5/40) of E.coli and 25.00%(5/20)of K.pneumoniae carried two types of CTXM gene. Twentythree isolates with TEM gene were TEM1;nineteen isolates with SHV gene included 6 isolates of genotype SHV1, 6 of SHV11, 5 of SHV12 and 2 of SHV25，only SHV12 was ESBLs gene,all these types were from K.pneumoniae; 66 isolates were with CTXM gene, the detection rate of CTXM14 among E.coli and K. pneumoniae was 45.00% and 35.00% respectively, CTXM55 were both 35.00%，CTXM15 was 20.00% and 15.00% respectively，CTXM3, CTXM65, CTXM101 and CTXM123 genotypes accounted for fewer percentage of CTXM genotype.ConclusionThe majority of ESBL gene in E. coli and K.pneumoniae in this hospital is CTXM, the next is SHV12. The most prevalent ESBL genotype is CTXM14. CTXM101 and CTXM123 are initially detected in Shandong province.