DNA is a versatile scaffold for molecular sensing in living cells, and various cellular applications of DNA nanodevices have been demonstrated. However, the simultaneous use of different DNA nanodevices within the same living cell remains a challenge. Here, we show that two distinct DNA nanomachines can be used simultaneously to map pH gradients along two different but intersecting cellular entry pathways. The two nanomachines, which are molecularly programmed to enter cells via different pathways, can map pH changes within well-defined subcellular environments along both pathways inside the same cell. We applied these nanomachines to probe the pH of early endosomes and the trans-Golgi network, in real time. When delivered either sequentially or simultaneously, both nanomachines localized into and independently captured the pH of the organelles for which they were designed. The successful functioning of DNA nanodevices within living systems has important implications for sensing and therapies in a diverse range of contexts.