Blastocystisis a common microeukaryotic intestinal parasite in humans and other animal hosts globally. However, no large-scale longitudinal study has ever been conducted forBlastocystis. To understand patterns of infection prevalence and subtype diversity and their relationship with host age, we have conducted the most comprehensive longitudinal study ofBlastocystisinfection ever performed. Dairy calves from a herd located in Maryland, USA, were followed from birth through 24 months of age, and 990 individual fecal samples from 30 calves were collected over the study period, representing three age groups (pre-weaned, post-weaned, and heifer). All samples were screened forBlastocystisvia PCR, and subtype determination was performed using next-generation amplicon sequencing. Associations between age group and infection status were assessed using logistic regression analyses.Blastocystisinfection prevalence increased with time, significant associations were observed between age groups and infection risk, and a cumulative prevalence of 100% was observed among the study population during the 24-month period. Thirteen previously reported subtypes (ST1-6, ST10, ST14, ST21, ST23–26) and one potentially novel subtype were observed. Diversity within ST10 supports the need for division of the subtype into new subtype designations. Associations between subtype and age group were explored, and relationships between subtypes and infection chronicity are described. While subtype diversity increased with age in the study population, distinct patterns of individual subtype prevalence and chronicity were observed, supporting the importance of subtype discrimination in studies of host infection and disease. The data from this study represent a significant advance in our understanding ofBlastocystisinfection dynamics within a single host population over time and can be used to inform future studies ofBlastocystisepidemiology in both humans and other animal hosts.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05795-0.