BK virus (BKV) reactivation arises from immunocompromised conditions and can produce a tubulointerstitial nephropathy (BKVN) in kidney transplant recipients (KTR). Approximately 5% of KTR develop BKVN, and about 45% of these lose their graft. Therefore, using molecular tools to test for BKV may be helpful in early detection. A series of 125 Spanish KTR, originating from a single transplant center, were studied in relation to BKV infection in the first post-transplant year. First, we carried out a urinary cytological study, looking for decoy cells as a possible marker of virus replication. Secondly, in all positive cytological samples and in some negative cytological samples (selected at random), we performed qualitative polymerase chain reaction (PCR) assays in serum and urine amplifying two different genome regions (LT and VP1). A transcription control region (TCR)-BK polymorphism sequence analysis was also performed in those BK PCR positive cases. Twenty-three of 125 (18.4%) KTR presented decoy cells in at least one urinary cytological sample. Molecular studies revealed that 10 of 125 (8%) KTR were BK PCR-serum positive cases (seven LT+/VP1- and three LT+/VP1+); and 13 of 40 (32.5%) KTR were BK PCR-urine positive cases (five LT+/VP1- and eight LT+/VP1+). When we compared PCR-urine and cytological results in 40 KTR, only 15% (six cases) revealed simultaneous positivity in both studies. In the context of clinical graft dysfunction, three patients demonstrated BK DNA presence in the renal biopsy. Finally, sequence analysis of the TCR was performed in 13 BK-PCR positive cases determining the AS, JL, WW, and WW-like viral variants. TCR sequence analysis, allows us to demonstrate the possible implication of the donor in BK infection studying four BK-PCR positive patients paired by donor.