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A comprehensive validation of data acquired by different myocardial perfusion imaging (MPI) systems was performed to evaluate contrast, self-attenuation properties, and perfusion detection capability. An anthropomorphic phantom with a myocardial insert and perfusion defect was used to simulate ^99mTc-tetrofosmin distribution. Different <i>MPI systems</i> were evaluated: a SPECT system with iterative reconstruction algorithms and resolution recovery (IRR) with/without scatter correction (SPECT-IRR-SC and SPECT-IRR), and a cardio-centric IQ SPECT/CT system with IRR, with/without scatter and attenuation corrections (IQ-IRR-SC-AC and IQ-IRR). The image quality was assessed through physical descriptors: the contrast between the left ventricular (<i>LV</i>) wall and <i>LV</i> inner chamber (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msub><mrow><mi>C</mi></mrow><mrow><mi>L</mi><mi>V</mi><mo>/</mo><mi>L</mi><mi>V</mi><mi>I</mi><mi>C</mi></mrow></msub></mrow></semantics></math></inline-formula>), intrinsic contrast (<i>IC</i>), and net contrast (<i>NC</i>). <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msub><mrow><mi>C</mi></mrow><mrow><mi>L</mi><mi>V</mi><mo>/</mo><mi>L</mi><mi>V</mi><mi>I</mi><mi>C</mi></mrow></msub></mrow></semantics></math></inline-formula> was found to be superior for IQ-IRR-SC-AC. The <i>IC</i> results showed non-uniformity of the signal intensity in the <i>LV</i> wall for the SPECT systems. The lowest <i>IC</i> values were obtained for IQ-IRR-SC-AC, except for septal position, where an underestimation of the signal intensity was revealed. The <i>NC</i> was found to be the highest for IQ-IRR-SC-AC and SPECT-IRR-SC. Additionally, for IQ-IRR-SC-AC, the <i>NC</i> increased in posterior and septal positions compared to IQ-IRR, enabling better perfusion detection capability over short-axis images. IQ-IRR showed performances comparable to SPECT-IRR. The characterization and evaluation perfusion detection capability of the <i>MPI systems</i> enabled the investigation of the systems’ performance and limitations.