Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK-deficient patients from 35 unrelated families. Twenty-nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in-frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N-terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three-dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme-linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype-to-phenotype correlation in PK deficiency as well as the enzyme's structure and function.