The increasing prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has been associated with the emergence of the CTX-M-producing sequence type 131 (ST131) pandemic clonal group, a member of the O25b serogroup and the B2 phylogenetic group. To assess the clonal spread of ESBL-producing E. coli in Japan, a regional surveillance programme was conducted.A total of 581 ESBL-producing clinical specimen E. coli isolates were collected between 2001 and 2010. Clonal groups, including ST131, D-ST405, D-ST393 and D-ST69, were determined using the PCR O type, phylogenetic grouping by triplex PCR, allele-specific PCR and multilocus sequence typing (MLST). A subset of clonal groups underwent PFGE.Among clonal strains, 215 isolates (37%) were identified as belonging to the ST131 group, 185 as B2-ST131-O25b (32%), 26 as B2-ST131-O16 (4%), 3 as B1-ST131-O25b (0.5%) and 1 as B2-ST131-O-non-typeable (0.1%). Forty-one isolates (7%) were identified as belonging to the D-ST405 clonal group, seven (1%) as D-ST69 and two (0.3%) as D-ST393. The B2-ST131-O16 clonal group was characterized by CTX-M-14 and a significantly lower ciprofloxacin resistance rate than the B2-ST131-O25b clonal group. The B2-ST131-O16 and B2-ST131-O25b clonal groups each made up a single PFGE cluster, with 65% similarity. The rate of ESBL-producing E. coli increased over the years (0.2% in 2001 to 9.7% in 2010) and corresponded to increases in the numbers of the B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups.The B2-ST131-O25b, B2-ST131-O16 and D-ST405 clonal groups have contributed to the spread of ESBL-producing E. coli in Japan.