PURPOSE: In Japan, the 7-valent pneumococcal vaccine (PCV7) was introduced in 2010 and, in 2013, the PCV7 was replaced with the 13-valent pneumococcal vaccine (PCV13). This study was conducted to investigate serotypes, antimicrobial resistance and prevalence of pilus islets in pneumococcal isolates from inpatients in a Japanese tertiary hospital. METHODOLOGY: From April 2011 to February 2016, 151 isolates [95 (18 children, 77 adults) and 56 (19 children, 37 adults) in the PCV7 and PCV13 periods, respectively] were collected. All isolates were serotyped using genetic methods and were tested for susceptibility to 18 antimicrobials. Unaltered penicillin-binding protein (PBP) genes, macrolide resistance genes and pilus islets were identified by PCR. RESULTS: Between the two periods, the prevalence of non-PCV13 serotypes was shown to increase from 50.0 to 78.9 % in children, and serotype 3 increased from 14.3 to 24.3 % in adults. Six of seven isolates from invasive diseases were assigned to non-PCV13 serotypes. Overall, multidrug resistance (MDR) was detected in 46.4 % of isolates, which included the dominant non-PCV13 serotypes 6E, 15A and 23A (prevalence≥75.0 %). gPRSP (three altered genes pbp1a, pbp2b and pbp2x) and macrolide resistance genes [erm(B) and/or mef(A/E)] were detected in 35.8 and 93.4 % of all isolates, respectively. Pilus islets [PI-1 (clade I, II and III) and/or PI-2] were found in 22.5 % (34/151) of isolates belonging to six different serotypes (19F, 23F, 19A, 6E, 15B and 35B) and 88.2 % (30/34) of these exhibited MDR. CONCLUSION: This study revealed the spread of MDR in several non-PCV13 serotypes and in isolates with pilus islets.