The purpose of this study was to define the incidence, dynamics, and profiles of anti-human leukocyte antigen antibodies (HLA-Abs) produced after kidney transplantation and their impact on graft outcome. A total of 72 first cadaver donor kidney recipients were prospectively monitored for the development of HLA-Abs using bead-based flow-cytometry assays (One Lambda FlowPRA tests). Sixteen recipients (22.2%) developed HLA-Abs after transplantation (class I, n = 7; class I+II, n = 6; class II, n = 3), in most cases (81.25%) within the first 2 weeks posttransplantation. A strong association between alloantibody presence and delayed graft function (Chi-square = 7.659, p < 0.01), acute rejection (Chi-square = 14.504, p < 0.001), chronic rejection (Chi-square = 12.84, p < 0.001), and graft loss (Chi-square = 20.283, p < 0.001) was found. Patients with higher alloantibody titers experienced acute rejections and even early graft loss, compared with those with lower titers for whom chronic rejections were more common. Immunologic complications occurred in recipients with both donor-specific and cross-reacting groups or non-donor-specific antibodies alone. A positive correlation (Pearson correlation, 0.245; p < 0.05) between HLA class I amino acid triplet incompatibility and alloantibody production was observed, mainly resulting from immunogenic triplotypes. Given the results obtained in this study, an alloantibody testing algorithm has been designed and implemented for routine monitoring and to define optimally the alloantibody reactivity in kidney transplant recipients.