OBJECTIVE The present study was designed to evaluate the molecular epidemiology of CTX-M producing Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli isolated from bloodstream infections at tertiary care hospitals in the State of Rio de Janeiro, Brazil. MATERIAL AND METHODS A total of 231 nonduplicate Enterobacteriaceae were isolated from five Brazilian hospitals between September 2007 and September 2008. The antimicrobial susceptibility testing was performed by disk diffusion method according to the Clinical Laboratory Standard Institute. Isolates showing resistance to third-generation cephalosporins were screened for ESBL activity by the double-disk synergy test. The presence of blaCTX-M, blaCTX-M-15 and blaKPC genes was determined by Polymerase Chain Reaction (PCR) amplification and DNA sequencing. The molecular typing of CTX-M producing isolates was performed by pulsed-field gel electrophoresis (PFGE). RESULTS AND DISCUSSION Ninety-three isolates were screened as ESBL positive and 85 (91%) were found to carry CTX-M-type, as follows: K. pneumoniae 59 (49%), E. cloacae 15 (42%), and E. coli 11 (15%). Ten isolates resistant for carbapenems in K. pneumoniae were blaKPC-2 gene positive. Among CTX-M type isolates, CTX-M-15 was predominant in more than 50% of isolates for K. pneumoniae, E. coli, and E. cloacae. PFGE analysis of CTX-M producing isolates showed the predominance of CTX-M-15 in 10 of 24 pulsotypes in K. pneumoniae, 6 of 13 in E. cloacae and 3 of 6 in E. coli. CTX-M-15 was also predominant among KPC producing isolates. In conclusion, this study showed that CTX-M-15 was circulating in Rio de Janeiro state in 2007-2008. This data reinforce the need for continuing surveillance because this scenario may have changed over the years.