Canine mammary tumors (CMTs) are considered a serious clinical problem in older bitches. Due to the high malignancy rate and poor prognosis, an early diagnosis is essential. This article is a summary of novel diagnostic techniques as well as the main biomarkers of CMTs. So far, CMTs are detected only when changes in mammary glands are clinically visible and surgical removal of the mass is the only recommended treatment. Proper diagnostics of CMT is especially important as they represent a very diverse group of tumors and therefore different treatment approaches may be required. Recently, new diagnostic options appeared, like a new cytological grading system of CMTs or B-mode ultrasound, the Doppler technique, contrast-enhanced ultrasound, and real-time elastography, which may be useful in pre-surgical evaluation. However, in order to detect malignancies before macroscopic changes are visible, evaluation of serum and tissue biomarkers should be considered. Among them, we distinguish markers of the cell cycle, proliferation, apoptosis, metastatic potential and prognosis, hormone receptors, inflammatory and more recent: metabolomic, gene expression, miRNA, and transcriptome sequencing markers. The use of a couple of the above-mentioned markers together seems to be the most useful for the early diagnosis of neoplastic diseases as well as to evaluate response to treatment, presence of tumor progression, or further prognosis. Molecular aspects of tumors seem to be crucial for proper understanding of tumorigenesis and the application of individual treatment options.