Chilling injury in cucumber (Cucumis sativus L.) is conditioned by maternal factors, and the sequencing of its chloroplast genome could lead to the identification of economically important candidate genes. Complete sequencing of cucumber chloroplast (cp)DNA was facilitated by the development of 414 consensus chloroplast sequencing primers (CCSPs) from conserved cpDNA sequences of Arabidopsis (Arabidopsis thaliana L.), spinach (Spinacia oleracea L.), and tobacco (Nicotiana tabacum L.) cpDNAs, using degenerative primer technologies. Genomic sequence analysis led to the construction of 301 CCSPs and 72 cucumber chloroplast-specific sequencing primers (CSSPs), which were used for the complete sequencing of cpDNA of Gy14 (155 525 bp) and 'Chipper' (155 524 bp) cucumber lines, which are, respectively, susceptible and tolerant to chilling injury (4 degrees C for 5.5 h) in the first leaf stage. Comparative cpDNA sequence analyses revealed that 1 sequence span (located between genes trnK and rps16) and 2 nucleotides (located in genes atpB and ycf1) differed between chilling-susceptible and -tolerant lines. These sequence differences correspond to previously reported maternally inherited differences in chilling response between reciprocal F1 progeny derived from these lines. Sequence differences at these 3 cpDNA sites were also detected in a genetically diverse array of cucumber germplasm with different chilling responses. These and previously reported results suggest that 1 or several of these sequences could be responsible for the observed response to chilling injury in cucumber. The comprehensive sequencing of cpDNA of cucumber by CCSPs and CSSPs indicates that these primers have immediate applications in the analysis of cpDNAs from other dicotyledonous species and the investigation of evolutionary relationships.