The cauliflower mosaic virus (CaMV) 35S promoter is known as the most frequently used promoter in plant biotechnology. Although it is widely considered to be a strong constitutive promoter exhibiting high transcriptional activity, the transcriptional stability of CaMV 35S has not been extensively studied. Using the model plant species Arabidopsis thaliana, this study aimed for a comprehensive expression analysis of two widely used plant transgenes, neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (EGFP), regulated by a double CaMV 35S promoter depending on the organ type, time of day, plant age, and in response to abiotic stress conditions. Quantitative real-time PCR (qRT-PCR) analysis revealed that the NPTII and EGFP transcript levels were markedly higher in the cotyledons, young leaves, and roots than in the inflorescences, stems, and adult leaves of three independent transgenic A. thaliana lines. The expression of NPTII and EGFP varied during the day and was elevated with the plant age. Drought and cold stress considerably affected the expression of the transgenes, while heat, high salinity, and wounding had no significant effect. This study shows that transgenes driven by a common constitutive promoter can exhibit marked variations in transcriptional activity depending on plant organ, physiological conditions, and in response to abiotic stress. Therefore, to ensure high and stable transgene activity, considerable attention should be given to the transgenic plant material and incubation conditions before harvesting the plant material.