Sooty blotch and flyspeck (SBFS) is a complex of >60 fungal species that blemish the surface of apple fruit in humid regions worldwide. Blemishes become visible in mid-to late summer, reducing the value of fresh fruit. To test the hypothesis that SBFS species appear on apples at characteristic times during the growing season, 22-37 apples were monitored weekly for appearance of SBFS colonies at each of three Iowa orchards in 2006 and seven orchards in 2007. Colonies were marked with colored pens to denote the date of appearance. After harvest and storage of apples at 4°C for 3 months, SBFS colonies on each fruit were counted and classified by morphology, and a representative subset of colonies with subtending peel was removed and pressed. Fungal DNA, extracted from colonies scraped from the surface of the peel, was amplified with primer pair ITS1-F/Myc1-R. Polymerase chain reaction products were digested with HaeIII, and fragment patterns were observed with gel electrophoresis and compared to a library of previously identified SBFS species. Colonies that could not be identified using band patterns were sequenced and compared to other species using BLAST. Sterile mycelia spp. RS1 and RS2 were the first to appear in all but one of the Iowa orchards surveyed where SBFS signs were observed. Dissoconium aciculare consistently appeared on fruit during the week prior to harvest, and additional colonies of this species appeared during storage. The species that were most prevalent in Iowa orchards were also the most abundant. Knowledge of species prevalence and chronology of appearance on apple fruit could lead to improved SBFS management strategies.