Genetic variation in cucumber (Cucumis sativus L. var. sativus) accessions from India was assessed by examining variation at 21 polymorphic isozyme loci. Forty-six accessions acquired by the U.S. National Plant Germplasm System (NPGS) before 1972 were compared with 146 accessions collected during a 1992 U.S.-India expedition to the states of Rajasthan, Madhya Pradesh and Uttar Pradesh, India. Isozymic profiles of these Indian accessions were also compared with 707 previously examined U.S. NPGS cucumber accessions. Two distinct groups (Group 1 and Group 2) were identified within accessions collected in 1992 (0.025 < P < 0.01). Variation at Ak-2, Fdp-2, Gr, Mdh-2, Mpi-1, Per, Pgm and Skdh was important in the detection of this difference. A previously unreported Pgm allele [Pgm (3)-105] was detected in accessions collected in a market in Pall, Rajasthan. Group 1 contained 37 (27 Madhya Pradesh + 10 Uttar Pradesh) accessions and Group 2 contains 102 (84 Rajasthan + 18 Madhya Pradesh) accessions. Seven accessions (5 Madhya Pradesh + 2 Rajasthan) were not associated with either group. The accessions 20664 (Tonk, Rahjasthan), 20666 (Jaipur, Rahjasthan), 20872 (Sehore, Madhya Pradesh), 20881 (Ashtok, Sehore, Madhya Pradesh) and 21026 (Bhatta, Dehra Dun, Uttar Pradesh) were heterozygous for at least nine loci and represent the genetic diversity within this collection. Isozymic variation in U.S. NPGS accessions acquired before 1972 differed significantly (P < 0.005) from those collected during 1992. All loci were important in the detection of this difference, except Ak-2, Pep-prp, and Pgd-2. When Indian accessions taken collectively (i.e., those acquired before 1972 and during 1992) were compared with an array of 707 C. sativus accessions examined previously, relationships between accessions grouped by country or subcontinent differed from those found in previous work.