Chilling damage can cause major reductions in cucumber (Cucumis sativus L.) yield. Cucumber plants can withstand a chilling event (i.e., tolerance and susceptibility), in which response is dictated primarily by maternally inherited plastid genomes or by the biparental contribution of a nuclear factor. To examine the modes of inheritance, exact reciprocal backcross cucumber populations (BC1–5), were created by crossing ‘Chipper’ (chilling-tolerant plastid, susceptible nucleus) and line North Carolina State University (NCSU) M29 (chilling-susceptible plastid, susceptible nucleus). These progeny and their parents were subjected to chilling stress [5.5 h at 4 °C in 270 μmol·m−2·s−1 photosynthetic photon flux (PPF) cool white lighting] at the first true-leaf stage. The chilling response of individuals possessing either NCSU M29 or ‘Chipper’ cytoplasm in any generational comparison was not significantly different (P > 0.05) from that of the maternal plastid source (susceptible or tolerant). Moreover, lines within a plastid type did not differ significantly (P > 0.05) in chilling response despite unequal nuclear contributions demonstrating the absence of nuclear additive or dosage effects originating in ‘Chipper’ or NCSU M29. Additionally, line NC-76, previously identified as a nuclear source of chilling tolerance, performed intermediate to ‘Chipper’ and NCSU M29 in chilling response under these stress conditions. The F1 progeny derived from crossing both BC5 plastidic response types (susceptible and tolerant) with NC-76 (paternal parent) performed comparable to their plastid donors and were significantly different (P < 0.0001) from one another despite their heterozygous nuclear nature resulting from the contribution of the nuclear chilling-tolerant factor contributed by NC-76. The response of tolerant and susceptible BC5 lines (i.e., ‘Chipper’ plastid in the NCSU M29 background and NCSU M29 plastid in ‘Chipper’ background, respectively) was reversible by crossing BC progeny with an alternate chilling-response plastid type. It is concluded that under these chilling conditions, plastid effects determine tolerance or susceptibility in the cucumber germplasms examined.