Background: Cancer-related fatigue (CRF) is reported to be associated with mitochondrial dysfunction. Hence, mitochondrial DNA (mtDNA) content, a biomarker of mitochondrial dysfunction, is hypothesized to correlate with the onset of CRF. This study aimed to evaluate the association between peripheral blood mtDNA content and CRF in patients receiving chemotherapy. Methods: This was a prospective cohort study. Early-stage breast cancer patients (Stages I to III) receiving anthracycline or taxane-based chemotherapy were recruited. CRF was assessed using the validated Multidimensional Fatigue Symptom Inventory-Short Form (MFSI-SF) at two time points: baseline (T1; prior to treatment) and 6 weeks after initiation of treatment (T2). Worsening of CRF was defined as ≥10% increase in the overall MFSI-SF score at T2. Peripheral blood mtDNA content was measured at both time points using real-time quantitative polymerase chain reaction. Multiple logistic regression was utilized to evaluate the association between mtDNA reduction and worsening of CRF, adjusting for age, anxiety, insomnia and other clinically important covariates. Results: A total of 91 patients [mean age (±SD): 51.3 (9.2) years; 81.3% Chinese; 63.3% receiving anthracycline-based chemotherapy] were recruited. Proportions of patients with worsening of CRF increased from the lower to the upper quartiles of mtDNA reduction (26.1%, 30.4%, 52.2%, and 59.1% in quartiles 1, 2, 3, and 4, respectively, P = 0.010 for trend). Reduction of mtDNA content was significantly greater among those with worsening of CRF compared to those without CRF [mean reduction (±SD): 16.3 (23.5) vs 6.0 (17.3), P = 0.018]. After adjusting for covariates, every 1-unit reduction of the mtDNA content was associated with a 4% increase risk for worsening of CRF (95% CI, 1%-8%; P = 0.016). Conclusions: This is the first study to show that reduction of mtDNA content in peripheral blood is associated with onset of CRF in patients receiving chemotherapy. Further validation studies are required to confirm the findings.