Background In previous work, we demonstrated the presence of hydroxyapetite (type II microcalcification), HAP, in triple negative MDA-MB-231 breast cancer cells. We used 18F-NaF to detect these types of cancers in mouse models as the free fluorine, 18F−, binds to HAP similar to bone uptake. In this work, we investigate other bone targeting agents and techniques including 99mTc-MDP SPECT and Osteosense 750EX FMT imaging as alternatives for breast cancer diagnosis via targeting HAP within the tumor microenvironment. Methods Thirteen mice were injected subcutaneously in the right flank with 106 MDA-MB-231 cells. When the tumor size reached ~0.6 cm3, mice (n = 9) were injected with ~37 MBq of 99mTc-MDP intravenously and then imaged one hour later in a NanoSPECT/CT or injected intravenously with 4 nmol/g of Osetosense 750EX and imaged 24 hours later in an FMT (n = 4). The imaging probe concentration in the tumor was compared to that of muscle. Following SPECT imaging, the tumors were harvested, sectioned into 10 µm slices, and underwent autoradiography or von Kossa staining to correlate 99mTc-MDP binding with HAP distribution within the tumor. The SPECT images were normalized to the injected dose and regions-of-interest (ROIs) were drawn around bone, tumor, and muscle to obtain the radiotracer concentration in these regions in units of percent injected dose per unit volume. ROIs were drawn around bone and tumor in the FMT images as no FMT signal was observed in normal muscle. Results Uptake of 99mTc-MDP was observed in the bone and tumor with little or no uptake in the muscle with concentrations of 11.34 ± 1.46 (mean ± SD), 2.22 ± 0.95, and 0.05 ± 0.04 %ID/cc, respectively. Uptake of Osteosense 750EX was also observed in the bone and tumor with concentrations of 0.35 ± 0.07 (mean ± SD) and 0.04 ± 0.01 picomoles, respectively. No FMT signal was observed in the normal muscle. There was no significant difference in the bone-to-tumor ratio between the two modalities (5.1 ± 2.3 for SPECT and 8.8 ± 2.2 for FMT) indicating that there is little difference in tumor uptake between these two agents. Conclusion This study provides evidence of the accessibility of HAP within the breast tumor microenvironment as an in vivo imaging target for bone-seeking agents. SPECT imaging using 99mTc-MDP can be rapidly translated to the clinic. FMT imaging using Osteosense 750EX is not currently approved for clinical use and is limited to animal research.