The pregnancy-associated hormones, progesterone (P4), pregnanediol-3-glucuronide (PdG), relaxin (RLN) and oestrone sulphate (E1S) in plasma, saliva, milk and urine of alpacas were measured in order to assess their potential use for pregnancy diagnosis. Samples were obtained from 36 female alpacas before mating and at different stages throughout pregnancy (confirmed by ultrasonography). The hormone concentrations were determined using enzyme immunoassays. Milk samples were also tested using a commercial on-farm P4 kit, designed for dairy cattle. Although the concentration of P4 in plasma, milk and urine, and the concentration of PdG in urine were significantly higher in pregnant than in non-pregnant alpacas, there was no difference in the concentrations of P4 or PdG in saliva. The on-farm milk P4 kit showed a sensitivity of 90 per cent for diagnosis of pregnancy and a specificity of 69 per cent for non-pregnancy. The concentration of RLN in plasma increased significantly after the second month, and concentration of E1S in plasma and urine during the last month of pregnancy, whereas, there were no significant differences in RLN or E1S concentrations in saliva and milk between pregnant and non-pregnant alpacas. Values of P4, RLN and E1S in plasma, and PdG and E1S in urine are comparable with the previous reports in alpacas and, therefore, can be confirmed as an indicator for pregnancy. This is the first study to include determination of pregnancy-associated hormones in the saliva and milk of alpacas. However, saliva seems to be unsuitable for pregnancy diagnosis in alpacas, whereas, P4 in milk, as well as PdG and E1S in urine, seem to be adequate tools for diagnosis.