The optimal approach to obtain an adequate graft for transplantation in patients with poor peripheral blood stem cell (PBSC) mobilization remains unclear. We retrospectively assessed the impact of different strategies of second-line stem cell harvest on the transplantation outcome of patients who failed PBSC mobilization in our institution. Such patients were distributed into three groups: those who proceeded to steady-state bone marrow (BM) collection (group A, n = 34); those who underwent second PBSC mobilization (group B, n = 41); those in whom no further harvesting was carried out (group C, n = 30). PBSC harvest yielded significantly more CD34+ cells than BM collection. Autologous transplantation was performed in 30, 23 and 11 patients from groups A, B and C, respectively. Engraftment data and transplantation outcome did not differ significantly between groups A and C. By contrast, group B patients had a faster neutrophil recovery, required less platelet transfusions and experienced less transplant-related morbidity, as reflected by lower antibiotics needs and shorter hospital stays. In conclusion, remobilization of PBSC constitutes an effective approach to ensure a rapid hematopoietic engraftment and a safe transplantation procedure for poor mobilizers, whereas unprimed BM harvest does not provide any clinical benefit in this setting.