Backscatter and attenuation coefficient estimates are needed in many quantitative ultrasound strategies. In clinical applications, these parameters may not be easily obtained because of variations in scattering by tissues overlying a region of interest (ROI). The goal of this study is to assess the accuracy of backscatter and attenuation estimates for regions distal to nonuniform layers of tissue-mimicking materials. In addition, this work compares results of these estimates for "layered" phantoms scanned using different clinical ultrasound machines. Two tissue-mimicking phantoms were constructed, each exhibiting depth-dependent variations in attenuation or backscatter. The phantoms were scanned with three ultrasound imaging systems, acquiring radio frequency echo data for offline analysis. The attenuation coefficient and the backscatter coefficient (BSC) for sections of the phantoms were estimated using the reference phantom method. Properties of each layer were also measured with laboratory techniques on test samples manufactured during the construction of the phantom. Estimates of the attenuation coefficient versus frequency slope, a0, using backscatter data from the different systems agreed to within 0.24 dB/cm-MHz. Bias in the a0 estimates varied with the location of the ROI. BSC estimates for phantom sections whose locations ranged from 0 to 7 cm from the transducer agreed among the different systems and with theoretical predictions, with a mean bias error of 1.01 dB over the used bandwidths. This study demonstrates that attenuation and BSCs can be accurately estimated in layered inhomogeneous media using pulse-echo data from clinical imaging systems. © Author(s) 2012.