Patterns of age differences between sexual partners – “age-mixing” – may partially explain the magnitude of HIV epidemics in Sub-Saharan Africa. However, evidence of age-disparity as a risk factor for HIV remains mixed. We used data from a socio-centric study of sexual behaviour in Malawi to quantify the age-mixing pattern and to find associations between relationship characteristics and age differences for 1,922 participants. Three age difference measures were explored as predictors of prevalent HIV infection. We found that for each year increase in male participant age, the average age difference with their partners increased by 0.26 years, while among women it remained approximately constant around 5 years. Women in the study had larger within-individual variation in partner ages compared to men. Spousal partnerships and never using a condom during sex were associated with larger age differences in relationships of both men and women. Men who were more than five years younger than their partners had 5.39 times higher odds (95% CI: 0.93–31.24) of being HIV-infected than men 0–4 years older. The relationship between HIV-infection and age-asymmetry may be more complex than previously described. The role that women play in HIV transmission should not be under-estimated, particularly in populations with large within-individual variation in partner ages.