Study of the ascidian collection at the National Museum of Nature and Science, Tsukuba, revealed some Japanese specimens of the non-native ascidian, Ascidiella aspersa (Müller, 1776), collected in 2007 from both mid-temperate Oominato, Mutsu Bay, in the northernmost part of Honshû, and from warm-temperate Ago Bay, Kii Peninsula, middle Honshû. These specimens were collected one year earlier than the previous earliest Japanese record from cool-temperate Funka Bay, Hokkaido. Mutsu Bay has an international port, which can be assumed to be the invasion gateway for this ascidian from abroad. On the other hand, this ascidian may have arrived at Ago Bay by domestic transport because all of the bay's ports are strictly for domestic use under governmental regulations. A comparison of the publicly available sequences for the 18S rRNA gene among this species and its allies suggested the possibility that it inhabited Korean waters as far back as the late 1990s, and it entered Japanese waters from Korea through an as yet unknown international port(s).