Crossing over, apart from its strictly genetic role, also performs a critical mechanical function, by bonding homologues in meiosis. Hence, it is responsible for an orderly reduction of the chromosome number. As such, it is strictly controlled in frequency and distribution. The well-known crossover control is the positive crossover interference which reduces the probability of a crossover in the vicinity of an already formed crossover. A poorly studied aspect of the control is the chromatid interference. Such analyses are possible in very few organisms as they require observation of all four products of a single meiosis. Here, we provide direct evidence of chromatid interference. Using in situ probing in two interspecific plant hybrids (Lolium multiflorum × Festuca pratensis and Allium cepa × A. roylei) during anaphase I, we demonstrate that the involvement of four chromatids in double crossovers is significantly more frequent than expected (64% vs. 25%). We also provide a physical measure of the crossover interference distance, covering ca. 30-40% of the relative chromosome arm length, and show that the centromere acts as a barrier for crossover interference. The two arms of a chromosome appear to act as independent units in the process of crossing over. Chromatid interference has to be seriously addressed in genetic mapping approaches and further studies.