Compared with clinical mastitis, the subclinical form of mastitis (SCM) is more common and thought to cause more economic losses to the dairy industry. The current study aimed to investigate the prevalence, risk factors of SCM, and effects on reproduction of dairy cows in major milk-producing areas of Sri Lanka. A total of 1,357 cows of selected farms in 3 regions were examined in the study. California Mastitis Test was conducted for individual cows, and a score of 2 or more for any quarter without any clinical symptoms and abnormalities in milk was considered as positive for SCM. Samples from infected animals were collected and subjected to bacteriological analysis. A pretested questionnaire was used to collect data on individual cows and herds. Risk factors associated with SCM were analyzed using binary logistic regression in generalized linear mixed models. The effect of SCM on calving to conception interval and days from calving to artificial insemination were analyzed by survival analysis using Cox's proportional hazards regression and Kaplan-Meier survival function estimates, respectively. A Poisson regression model was run to determine the effect of SCM on number of artificial inseminations per conception. The prevalence of SCM was 57.5, 11.8, and 45.5% in the regions A, B, and C, respectively. The most common pathogen was Staphylococcus aureus, with 87.1, 56.5, and 92.3% in the regions A, B, and C, respectively. Logistic regression analysis showed that parity, farming system, milking area, region, and herd significantly affect the prevalence of SCM. Subclinical mastitis during the pre-breeding period was associated with 14% increase in the chance of having a greater number of artificial inseminations per conception. Likewise, median days from calving to artificial insemination was longer in cows with SCM compared with normal cows (79 and 64 d, respectively). Therefore, SCM affected the inseminated proportion of studied cows. However, SCM was not associated with the calving to conception interval. The results revealed that the cow factors and milk hygiene play a significant role in the prevalence of SCM.