bla(TEM-1) expression results in penicillin resistance, whereas expression of many bla(TEM-1) descendants, called extended-spectrum beta-lactamases (ESBLs), results simultaneously in resistance to penicillins and extended-spectrum cephalosporins. Despite the expanded resistance phenotypes conferred by many ESBLs, bla(TEM-1) is still the most abundant bla(TEM) allele in many microbial populations. This study examines the fitness effects of the two amino acid substitutions, R164S and E240K, that have occurred repeatedly among ESBL bla(TEM-1) descendants. Using a single-nucleotide polymorphism-specific real-time quantitative PCR method, we analyzed the fitness of strains expressing bla(TEM-1), bla(TEM-10), and bla(TEM-12). Our results show that bacteria expressing the ancestral bla(TEM-1) allele have a fitness advantage over those expressing either bla(TEM-10) or bla(TEM-12) when exposed to ampicillin. This observation, combined with the fact that penicillins are the most prevalent antimicrobials prescribed worldwide, may explain why bla(TEM-1) has persisted as the most frequently encountered bla(TEM) allele in bacterial populations.