Chinese Spring (CS) wheat (Triticum aestivum L.) is commonly used in genetic research including cytogenetic analysis, molecular mapping and germplasm development. Aneuploid lines of alien chromosomes in the CS background have been used in studies with diverse objectives. Thousands of genomic and complementary DNA sequences from expressed sequence tag (EST) libraries of biotic- and abiotic-stressed tissues are publicly available from CS. Gene expression analysis of salt-tolerant wheat lines, W4909 and W4910, compared with the CS common wheat background led to the discovery of several expressed sequences that were absent in the CS accession held in our laboratory. A survey of 20 CS accessions from 13 laboratories using the polymerase chain reaction with gene-specific primers for eight salt-responsive genes resulted in amplification success ranging from 15 to 100%. Amplified fragment length polymorphism analysis showed that 99% of the genetic variation was among the accessions while the remaining 1% was within the accessions. A neighbour-joining phylogram showed that four of the five CS accessions from the International Maize and Wheat Improvement Center (CIMMYT) grouped with the salt-tolerant wheat cultivar Yecora Rojo while the remaining 16 CS accessions had limited genetic differences. Thus, variation exists among these highly self-pollinating CS sources, suggesting that appropriate consideration should be taken when using CS accessions to conduct molecular and genetic analyses.