One of the most damaging invasive insects is the red palm weevil Rhynchophorus ferrugineus (RPW). It considers the most damaging insect pest of date palm. In the present study, in vivo and in vitro genetic differences among RPW females from five different regions of Egypt were investigated through RAPD markers and the partial sequence of CO1 gene. Ten primers were used in RAPD analysis. 46 of the 60 produced fragments with a ratio of 76.67% were polymorphic and 14 fragments with a ratio of 23.33% were monomorphic between the samples of the different studied regions. There was no difference between in vivo and in vitro four samples collected from the five studied geographic regions by using RAPD analysis. The multi-alignment between in vivo and in vitro samples of CO1 partial sequence of studied populations revealed the absence of any nucleotide differences between the samples of the same region. The neighbor-joining tree between the studied RPW populations and the closed sequences from Gen Bank placed them in two different clusters with different haplotypes from Egypt and the Kingdom of Saudi Arabia. According to CO1 gene partial sequence and RAPD analysis results, it can be concluded that there are slight effects of cell culture media compositions on the DNA of cells. The study results suggest that cell culture methods can be used in biological control studies instead of in vivo ones. Also, there may be more than one haplotype of RPW in Egypt and it may be introduced from the same or different sources.