OBJECTIVE To compare endophthalmitis rates after 20-gauge versus 23-gauge versus 25-gauge pars plana vitrectomy (PPV) in 2007-2008, and compare the rates with those of 2005-2006. METHODS Multicenter study including all patients who developed endophthalmitis following PPV performed by any of the authors during 2005-2008, and all patients who developed endophthalmitis following PPV at Penn State College of Medicine and Bascom Palmer Eye Institute during 2005-2008. The endophthalmitis rates after 20-gauge, 23-gauge and 25-gauge PPV during 2007-2008 were compared to those from 2005-2006. RESULTS The endophthalmitis incidence during 2007-2008 was 1/4,403 (0.02%) for 20-gauge PPV, 1/3,362 (0.03%) for 23-gauge PPV, and 1/789 (0.13%) for 25-gauge PPV. There is no significant difference among these rates between any two of the three groups. Compared with the endophthalmitis rates among the same group of surgeons during 2005-2006, the 2007-2008 endophthalmitis rates following 20-gauge and 23-gauge PPV were stable, and the rate following 25-gauge PPV was marginally lower (p = 0.056; odds ratio = 0.15; 95% CI: (0.003, 1.03)). CONCLUSIONS There was no significant difference in the 2007-2008 rates of endophthalmitis following 20-gauge versus 23-gauge versus 25-gauge PPV; among the same group of surgeons, the 2007-2008 rate of endophthalmitis following 25-gauge PPV was marginally lower than the 2005-2006 rate.