The development of Elaeis guineensis chromosome-arm specific markers is required for linking the genetic, sequence and chromosomal maps, valuable for comparative studies, hybridisation and breeding. Here, we aimed to develop strategies exploiting assembled short shotgun-sequence reads to identify markers for in situ hybridisation to identify arms and large-scale organisation of pseudo-chromosome 1. The conserved putative repetitive DNA sequence found through informatics analysis showed an extra intercalary band in one arm of pseudo-chromosome 1 in fluorescence the in situ hybridisation result. The results are a basis for establishing a North-South orientation of E. guineensis pseudo-chromosome. Furthermore, the ability of the newly developed markers for distinguishing both Elaeis species with in situ hybridisation showed their utility in identifying Elaeis hybrids.