Chlamydia spp. are obligate intracellular gram-negative bacteria that cause a wide range of significant diseases in humans and animals worldwide, resulting in significant economic losses. Chlamydial infection in cattle has been reported in many countries including China. However, there has been no survey of chlamydial infection of dairy cattle in Guangzhou, southern China. The objective of the present investigation was to examine the chlamydial seroprevalence in dairy cattle in Guangzhou, subtropical southern China by using an indirect hemagglutination assay (IHA). The overall seroprevalence of chlamydial infection in dairy cattle was 7.25% (29/400). Greater than or equal to eight-yr-old dairy cattle had the highest seroprevalence (10.34%), followed by those that were ≥ 6 years old or < 7 years old dairy cattle (10.20%), although there were no statistically significant differences among different groups (P > 0.05). Dairy cattle with 5 pregnancies had the highest seroprevalence (10.81%). These results indicate that chlamydial infection was present in dairy cattle in Guangzhou, subtropical southern China, and integrated strategies and measures should be executed to control and prevent chlamydial infection and disease outbreak in the study region.