ABSTRACT Extended-spectrum β-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV β-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla _SHV-2a gene and seven strains carried the bla _SHV-12 gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla _SHV-11 gene and one strain carried the non-ESBL bla _SHV-1 gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla _SHV-2a or bla _SHV12 gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed “first-nucleotide change,” involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla _SHV-2a from strains carrying bla _SHV-12 . In addition, this method was used to demonstrate an association between the relative copy numbers of bla _SHV genes in individual strains and the levels of antibiotic resistance.