Chloroplast RNA splicing is usually studied by complicated methods, such as Northern blot hybridization, RNAse protection, or primer extension assays. The use of a simpler RT-PCR technique frequently results in the underestimation of unspliced pre-mRNA levels. Five protein-coding genes from the maize plastome were studied to analyze which factors can lead to an apparent reduction in the pool of intron-containing transcripts compared to mature RNA. It was found that the accurate determination of the unspliced RNA level should take into account the DNase inactivation mode, the cDNA synthesis temperature, and the type of DNA polymerase. For one gene, the portion of unspliced RNAs decreased with increasing number of PCR cycles. It was found that intron-containing and intron-free strands of amplicons can form heteroduplexes after PCR. A simple and effective technique of investigating chloroplast spliced mRNA and unspliced pre-mRNA is suggested based on the obtained data.