A multilocus VNTR analysis (MLVA) system for detection of tandem repeats across the whole genome of Chlamydophila psittaci has been developed. Twenty selected genetic loci were initially tested on 9 avian reference strains including representatives of all major serotypes (A to F). Thereafter, 8 loci were retained for a more complete study performed on over 150 C. psittaci isolates from different bird species and geographical origins. Comparative analysis of the MLVA results and those obtained from currently available methods including serotyping and/or ompA sequencing indicate that the MLVA system provides an additional level of discrimination, with 20 distinct patterns identified to date. The newly developed MLVA system therefore provides a highly sensitive, high resolution test for the differentiation of C. psittaci isolates from different origins that is suitable for molecular epidemiological studies.