Ascaris roundworm isolates from Japan and central Europe were examined by sequencing analyses to better understand geographically induced nucleotide variation and genotype distribution according to host. Three well-supported clusters (denoted as A, B, C) were identified by generating cox1 sequences of mtDNA from these regions. Among 5 pig isolates collected in eastern Honshu, Japan, in 2010, 3 carried DNA characteristics for cluster A and 2 corresponded with the characteristics of cluster B. The sequence of the human isolate JH1 from north-central Honshu, fixed in formalin since 1972, conformed to the characteristics of cluster A. Differential analysis of ribosomal ITS1 region revealed the JH1 isolate sequence profile of Ascaris lumbricoides. Cluster C, which was the most distinguish cluster, was formed by reference Slovak isolates and has been so far found almost exclusively in European pigs. A fluctuating prevailing distribution of A and B lineages in human and pig hosts in different territories of the world and the global distribution of several haplotypes indicate their establishment before secondary differentiation in a given region due to host affiliation. The protocol established for DNA isolation from formalin-fixed specimens using the modified procedure with the Qiagen extraction set can be used as a tool for retrospective studies in ascarid helminths when only archival specimens are available.