Recognition of myxomatosis is usually based on clinical symptoms, but amyxomatous cases of the disease require the use of laboratory methods. Nowadays PCR assays are routinely employed for detection of MYXV DNA, but none of them have had their diagnostic usefulness conclusively confirmed through validation. The aim of the study was the development and validation of a PCR with an internal amplification control (IAC) for intravital and postmortem detection of viral DNA of myxoma virus. To avoid false negative results a chimeric internal amplification control (IAC) was prepared and incorporated into the PCR and amplified by the same primer set as the target DNA (M071L). The optimal concentration of particular ingredients in the PCR mixture (including IAC concentration and volume of DNA sample) was determined. To minimize the risk of amplicon carry-over contamination, uracil N-glycosylase was added to the reaction. Before proper validation the robustness of the IACPCR was verified. Validation of the method encompassed the following parameters: the analytical and diagnostic specificity (ASp, DSp) and sensitivity (ASe, DSe) of the assay, repeatability, and intra-laboratory reproducibility. The assay LOD was established at 2 TCIU of the virus particles/0.2 ml tissue homogenate with a 100% capacity to detect different MYXV strains (ASp). The method was characterized by good DSp of 0.955 (0.839–0.999 CI) and DSe of 0.976 (0.914–1.00 CI). In addition, it was repeatable and reproducible and confirmed its suitability for the detection of MYXV in clinical material. The IAC-PCR developed meets OIE validation requirements for virological methods and can be used in diagnostic or epidemiological studies of rabbit myxomatosis.