Isolation of novel types of human adenovirus D (HAdV-53, -54, and -56) from keratoconjunctivitis patients has been reported since 2008. We examined the molecular epidemiology of HAdV-D strains using 39 field isolates collected from epidemic keratoconjunctivitis (EKC) patients from 2001 to 2010 in the province of Osaka, Japan. The molecular types were analyzed by sequencing partial penton base gene, loop 1 in the hexon, and complete fiber genes. Of the 39 isolates, the majority were HAdV-19 (14/39, 35.9%) and -37 (13/39, 33.3%), followed by -53 (4/39, 10.3%) and -54 (8/39, 20.5%). Analysis of annual distribution showed that HAdV-19 and -37 were detected before 2004, whereas HAdV-53 and -54 were first identified in 2001 and 2003, respectively, and persistently detected during the study period. It is noted that both HAdV-53 and -54 isolates were misclassified by the serological method. Altogether, the molecular analysis elucidated the epidemiology of HAdV-D and presence of novel types from the early 2000s in Osaka. Further genetic analysis of serologically classified HAdV-D isolates may provide insights into the epidemiology of EKC.