Measurement of cytokines in the lower female genital tract offer insight into risk for HIV infection and reproductive complications. However, few studies have systematically compared mucosal collection methods or whether collection order matters. We compared longitudinal cytokine profiles in matched genital samples collected from women living with HIV using menstrual cup (MC), endocervical swabs (ECS) and swab-enriched cervicovaginal lavage (eCVL). Samples were collected at enrollment [MC:ECS:eCVL], 3-months (ECS:eCVL:MC) and 6-months (eCVL:MC:ECS) and concentrations of 28 cytokines determined by Luminex. Cytokine clustering was assessed using Principle Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLSDA) and factor analysis. Generally, higher cytokine concentrations were detected in MC samples, followed by ECS and eCVL, irrespective of study visit or sampling order. Factor analysis and PCA identified ECS to be inferior for measuring regulatory cytokines and IP-10 than eCVL or MC. Although concentrations differed, the majority of cytokines correlated between methods. Sampling order influenced cytokine concentrations marginally, and cytokines clustered more strongly by method than study visit. Variance in profiles was lowest in MC, suggesting greater consistency of sampling compared to other methods. We conclude that MC sampling offered advantages over other methods for detecting cytokines in women, with order marginally influencing profiles.