In this study, we have analyzed Cucumber mosaic virus (CMV)-resistant transgenic hot pepper (Capsicum annuum) plants harboring coat protein gene (CMVP0-CP) of the virus as a framework for molecular and cytogenetic assessment. PCR, Southern blot, RT-PCR, and Western blot techniques were applied to identify the existence and expression of the transgene in two independent transgenic hot pepper events in $T_4$ generation, H15 and B20. Fluorescence in situ hybridization (FISH) was also conducted to detect physical location of the transgene on the chromosome of the plants. The amplified products of the inserted genes, 35S promoter with CMVP0-CP, CMVP0-CP, and NPT II were obtained from the genomic DNA of the transgenic hot pepper lines by PCR. A single band, indicative of a single copy insertion, was observed by Southern blot analysis. Transcription of the transgene was confirmed from RT-PCR fragments CMVP0-CP and NPT II genes in transgenic plants. Translated protein of the target gene, CMV-CP, was detected in the transgenic hot pepper events but not in nontransgenic control hot pepper lines by Western blot analysis. FISH analysis of transgenic line H15 revealed that the transgene was physically located at the sub-terminal portion of the long arm of chromosome d where the 5S rDNA locus is positioned. Conclusively, we observed that transgenes were maintained stably in $T_4$ generation of transgenic hot peppers via cytogenetic investigation as well as molecular detection. These results can be useful in future studies conducted to evaluate breeding techniques and to assess genetically modified hot peppers.