The PK‐LR gene has been studied in 12 unrelated patients with red cell pyruvate kinase deficiency and hereditary nonspherocytic haemolytic anaemia (CNSHA). The entire codifying region of the R‐type PK gene and the flanking intronic regions were analysed by single‐stranded conformation polymorphism (SSCP) followed by direct sequencing of abnormal DNA. 10 different mutations were identified in 22/24 alleles at risk. Eight of these were missense mutations that caused the following single amino acid changes: G514C (172Glu‐Gln), G1010A (337Arg‐Gln), G1015C (339Asp‐Gln), T1070C (357Ile‐Thr), C1223T (408Thr‐Ile), G1291A (431Ala‐Thr), C1456T (486Arg‐Trp) and G1595A (532Arg‐Gln). Two were nonsense mutations: G721T (241Glu‐Stop) and C1675T (559Arg‐Stop). 7/22 alleles demonstrated the same C1456→ T mutation. The study of the polymorphic site at nucleotide (nt) 1705 performed in all cases disclosed a 1705 C/C mutation in 10 and a 1705 A/C mutation in three. This is the first report on the presence of several different L‐type PK gene mutations within Spanish population. Furthermore, from the PK gene mutations found, six were unique and not previously described (1015C, 1070C, 1223T, 1291A, 1595A and 1675T) and one (C1456T) seems to be predominant in Spain. Interestingly, no case with the 1529A mutation commonly found in Northern European populations was present here.