Milk production is prioritized in Rwanda as it represents an essential food source for many families, therefore, higher milk yields are desired. Subclinical mastitis (SCM) is one reason for low milk yield but SCM has not been well studied in Rwanda. The objective of this study was to evaluate prevalence, causative udder pathogens, and cow and herd risk factors associated with SCM in dairy cows linked to milk collection centers (MCC) in Rwanda. Screening with the California Mastitis Test (CMT) was done on 572 cows from 404 herds from 2 MCCs in each of 4 provinces. Milk from udder quarters with CMT score ≥ 3 (scale 1–5) was sampled for bacteriological analyses by culture and a final identification with matrix-assisted laser desorption ionization time of flight mass spectrometry. The prevalence of SCM was 37.3% at the quarter level and 62.0% at cow level. There was a significant (P ≤ 0.05) difference in SCM prevalence among the MCCs, with highest prevalence in the 2 MCCs in the northern province. Bacteria were isolated in 66.3% of the cultured milk samples, whereas culture-negative and contaminated samples were 30.7% and 2.99%, respectively. Non-aureus staphylococci (NAS) and S. aureus were the most prevalent pathogens, representing more than half of all bacteriological findings. Staphylococcus chromogenes and S. epidermidis were the most prevalent NAS identified. Beta lactamase production was present in 65.7% of all staphylococci isolates. The risk factor analysis indicated that an increased stage of lactation, dirty udder and legs, no calf suckling the dam and not feeding concentrates were associated with increased odd ratio of SCM in cows. Absence of foremilk stripping was the only factor associated with S. aureus infection. High SCM prevalence could affect yield and milk quality at the MCC level. Identified pathogens suggest a possible high rate of within quarter or cow infection transmission and low cure rate against penicillin. Control of the identified risks factors and biosecurity by adjustments in practices, and farmer training could contribute to lowering SCM in Rwanda